Methods for raising pre-adult anadromous fish

ABSTRACT

The invention relates to methods, compositions and kits for improving the raising of pre-adult anadromous fish, or preparing pre-adult anadromous fish for transfer to seawater. The methods involve adding at least one Polyvalent Cation Sensing Receptor (PVCR) modulator to the freshwater in an amount sufficient to increase expression and/or sensitivity of at least one PVCR; and adding feed for fish consumption to the freshwater, wherein the feed comprises an amount of NaCl sufficient to contribute to a significantly increased level of the PVCR modulator in serum of the pre-adult anadromous fish.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/270,876, filed Oct. 11, 2002 now U.S. Pat. No. 6,655,318, which is adivisional of U.S. application Ser. No. 09/687,477, filed Oct. 12, 2000,now U.S. Pat. No. 6,463,883. The entire teachings of the aforementionedapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

In nature, many anadromous fish live most of their adulthood inseawater, but swim upstream to freshwater for the purpose of breeding.As a result, anadromous fish hatch from their eggs and are born infreshwater. As these fish grow, they swim downstream and gradually adaptto the seawater.

Fish hatcheries have experienced difficulty in raising these types offish because the window of time in which the pre-adult fish adapts toseawater (e.g., undergoes smoltification) is short-lived, and can bedifficult to pinpoint. As a result, these hatcheries experiencesignificant morbidity and mortality when transferring anadromous fishfrom freshwater to seawater. Additionally, many of the fish that dosurvive the transfer from freshwater to seawater are stressed, andconsequently, experience decreased feeding, and increased susceptibilityto disease. Therefore, these anadromous fish often do not grow wellafter they are transferred to seawater.

The aquaculture industry loses millions of dollars each year due toproblems it encounters in transferring pre-adult anadromous fish fromfreshwater to seawater. Hence, a need exists to improve methods involvedin transferring pre-adult anadromous fish to seawater. A further needexists to increase survival and growth, and reduce stress, of pre-adultanadromous fish that have been transferred to seawater.

SUMMARY OF THE INVENTION

The present invention relates to methods for improving the raising ofpre-adult anadromous fish or preparing these fish for transfer toseawater by increasing expression of a receptor, referred to as thePolyvalent Cation Sensing Receptor (PVCR). The expression and/orsensitivity of the PVCR is increased by subjecting the pre-adultanadromous fish to at least one modulator of the PVCR. The pre-adultanadromous fish are subjected to the modulator when it is added to theirfreshwater environment, and optionally, to the feed. The inventionencompasses adding at least one PVCR modulator to the freshwater, andadding feed for fish consumption to the freshwater. The feed containssodium chloride (NaCl) and, optionally, at least one PVCR modulator inan amount to contribute to a significantly increased level of the PVCRmodulator in the serum of the pre-adult anadromous fish. Increasedexpression and/or sensitivity of the PVCR is maintained until the fishare ready to be transferred to seawater. The pre-adult anadromous fishcan be maintained in the freshwater having at least one PVCR agonistuntil they are ready to be transferred to seawater.

In one embodiment of the invention, pre-adult anadromous fish (e.g.,salmon, trout and arctic char) are prepared for transfer from freshwaterto seawater by adding PVCR agonists, such as calcium and magnesium tothe freshwater, and adding feed for fish consumption having betweenabout 1% and about 10% NaCl by weight (e.g. between about 10,000 mg/kgand 100,000 mg/kg) to the freshwater. The amount of calcium added to thefreshwater is an amount sufficient to bring the concentration up tobetween about 2.0 mM and about 10.0 mM, and the amount of magnesiumadded is an amount sufficient to bring the concentration up to betweenabout 0.5 mM and about 10.0 mM. The feed can optionally include a PVCRagonist, such as an amino acid. A particular amino acid that can beadded is tryptophan in an amount between about 1 gm/kg and about 10gm/kg. The present invention also includes, optionally, exposing thepre-adult anadromous fish to a photoperiod. Preferably, the photoperiodis continuous (e.g., for a continuous period of between about 12 hoursand about 24 hours in a 24 hour period).

Additional embodiments of the invention include methods of increasing orimproving food consumption before and/or after seawater transfer,increasing growth, increasing survival and/or reducing mortality,reducing osmotic damage, transferring parr (e.g., between about 10 andabout 60 grams) to seawater, and transferring a pre-adult anadromousfish to seawater having a temperature of about 14° C. to about 19° C.These methods are performed by adding at least one PVCR modulator to thefreshwater, subjecting or exposing the pre-adult anadromous fish to atleast one PVCR modulator, or introducing the pre-adult anadromous fishto freshwater having at least one PVCR modulator, in an amountsufficient to increase expression and/or sensitivity of the PVCR. Themethods also involve adding feed having between about 1% and about 10%NaCl by weight to the freshwater and transferring the pre-adultanadromous fish to seawater.

In other embodiments, the invention encompasses detection assays ormethods of determining whether a pre-adult anadromous fish, that aresubjected to at least one PVCR modulator and are fed with feed havingbetween about 1% and about 10% NaCl by weight, are ready for transfer toseawater, by assessing the amount of PVCR expression in the pre-adultanadromous fish. An increased level of expression and/or sensitivity, ascompared to a control (e.g., PVCR expression from a fish not subjectedto a PVCR modulator), indicates that the pre-adult anadromous fish areready for transfer to seawater. In a preferred embodiment, the assayincludes contacting an anti-PVCR antibody with a sample (e.g., gill,skin, intestine, urinary bladder, kidney, brain or muscle) underconditions sufficient for the formation of a complex between theantibody and the PVCR; and detecting the formation of the complex. Inanother embodiment the assay relates to hybridizing a nucleic acidsequence having a detectable label to the nucleic acid sequence of thePVCR of a sample taken from the pre-adult anadromous fish and detectingthe hybridization.

In yet another embodiment, the present invention relates to variouscompositions and mixtures. In particular, the invention pertains to anaquatic food composition having a concentration of NaCl between about10,000 mg/kg and 100,000 mg/kg (e.g., about 12,000 mg/kg). The aquaticfood composition can optionally include a PVCR modulator (e.g.tryptophan in an amount between 1 gm/kg and 10 gm/kg).

The invention also embodies an aquatic mixture for providing anenvironment to improve the raising of pre-adult anadromous fish. Themixture includes at least one PVCR modulator. An example of such amixture is a calcium source, that when added to freshwater, provides aconcentration of between about 2.0 mM and about 10.0 mM; and a magnesiumsource, that when added to freshwater, provides a concentration ofbetween about 0.5 mM and 10.0 mM.

In yet another embodiment, the present invention relates to kits. Inparticular, the invention embodies kits for improving the raising ofpre-adult anadromous fish, that includes a PVCR modulator for additionto the freshwater and an aquatic food composition, as described herein.In another embodiment, the invention includes kits for determiningwhether a pre-adult anadromous fish are ready for transfer to seawater,after being subjected to at least one PVCR modulator and feed havingbetween about 1% and about 10% NaCl by weight. The kit includes eitheran anti-PVCR antibody, and a solid support; or a nucleic acid sequencehaving a detectable label that can hybridize to nucleic acid of anaquatic PVCR.

Surprisingly, it has been discovered that increased expression and/oraltering the sensitivity of the PVCR allows these pre-adult anadromousfish to better adapt to seawater. Until the discovery of the presentinvention, the aquaculture industry was unable to transfer the pre-adultanadromous fish to seawater without subjecting the fish to stress, deathand/or disease. Unlike this practice, carrying out the steps of theinvention increases the expression and/or alters the sensitivity of thePVCR and allows for transfer of the pre-adult anadromous fish toseawater with minimal or no stress, death and/or disease, andunexpectedly provides several benefits, such as increased growth and theability to transfer these fish to water having higher temperatures, asfurther described herein. The present invention results in one or moreof the following advantages in transferring pre-adult anadromous fish toseawater: a reduction in mortality; improvement in feeding; an increasein growth; a decrease in the amount of diseased fish; and/or a reductionin osmotic shock. The present invention also allows for earlierharvesting of the fish with increased flexibility in producing fish yearround. Additionally, the methods of the present invention can result insignificant cost savings for fish hatcheries.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating the partial nucleotide (SEQ ID NO:1)and amino acid (SEQ ID NO:2) sequences of the polyvalent cation-sensingreceptor (PVCR) of Atlantic salmon (Salmo salar).

FIG. 2 is a diagram illustrating the partial nucleotide (SEQ ID NO:3)and amino acid (SEQ ID NO:4) sequences of the PVCR of arctic char(Salvelinus alpinus).

FIG. 3 is a diagram illustrating the partial nucleotide (SEQ ID NO:5)and amino acid (SEQ ID NO:6) sequences of the PVCR of rainbow trout(Onchorhynchus mykiss).

FIGS. 4A–B are diagrams illustrating the alignment of the amino acidssequences for shark kidney cation receptor (“SKCaR”) (SEQ ID NO: 18),salmon (SEQ ID NO:2), arctic char (SEQ ID NO:4) and rainbow trout (SEQID NO:6).

FIG. 5 is a schematic diagram illustrating industry practice for salmonaquaculture production, prior to the discovery of the present invention.The diagram depicts key steps in salmon production for S0 (75 gram) andS1 (100 gram) smolts. The wavy symbol indicates freshwater while thebubbles indicate seawater.

FIG. 6 is a graphical representation illustrating length (cm) and weight(gm) of APS Process I Smolts 50 days after ocean netpen placement. APSProcess I smolts had an average weight of 76.6 gram when placed seawaterand were sampled after 50 days. APS Process I is defined is Example 2.

FIG. 7 is a graphical representation illustrating length (cm) and weight(gm) of representative APS Process I smolts prior to transfer toseawater.

FIG. 8 is a graphical representation illustrating length (cm) and weight(gm) of APS Process I smolts before transfer, and mortalities.

FIG. 9 is a three dimensional graph illustrating the survival over 5days of Arctic Char in seawater after being maintained in freshwater,APS Process I for 14 days, and APS Process I for 30 days.

FIG. 10 is a graphical representation illustrating the length (cm) andweight (gm) of St. John/St. John APS Process II smolts prior to seawatertransfer. APS Process II is defined in Example 2.

FIGS. 11A and 11B are graphical representations illustrating weight (gm)and length (cm) of APS Process II smolt survivors and mortalities 5 daysafter transfer to seawater tanks, and 96 hours after transfer to oceannetpens.

FIG. 12 is a graphical representation showing weight (gm) and length(cm) of APS Process II smolt mortalities after 5 days after transfer toocean netpens.

FIGS. 13A–G are photographs of immunocytochemistry of epithelia of theproximal intestine of Atlantic Salmon illustrating PVCR localization andexpression.

FIG. 14 is a photograph of a Western Blot of intestinal tissue fromsalmon maintained subjected to APS Process I for immune (lane markedCaR, e.g., a PVCR) and preimmune (land marked preimmune) illustratingPVCR expression.

FIG. 15 is a photograph of a Western Blot of intestinal tissue fromtrout fingerlings for immune (lane marked CaR, e.g., a PVCR) andpreimmune (lane marked preimmune) illustrating PVCR expression.

FIGS. 16A–H are photographs of immunocytochemistry of epithelia ofproximal intestine of rainbow trout using anti-PVCR antiserumillustrating PVCR localization and expression.

FIG. 17 is a photograph of a Western Blot comparing levels of PVCR offish in freshwater, water having calcium and magnesium, and seawater,illustrating PVCR expression.

FIGS. 18A–C are photographs of immunolocalization of the PVCR in theepidermis of salmon illustrating PVCR localization and expression.

FIG. 19 is a schematic drawing illustrating adaptive changes of fish inseawater and in freshwater.

FIG. 20 is a graphical representation of serum calcium concentrations(mM) over time in rainbow trout subjected to transfer to either seawateror water mixture of the present invention. All data points represent aleast 5 independent determinations mean±standard deviation from a singlerepresentative experiment.

FIG. 21 is a graphical representation showing increases in serum calciumconcentrations (mM) over time induced by feeding trout maintained in awater mixture (3 mM calcium, 1 mM magnesium) and a standard freshwaterpelleted diet containing additional 1% sodium chloride (w/w).

FIGS. 22A and 22B are graphical representations of alterations in serumcalcium (FIG. 22A) and sodium (FIG. 22B) after seawater transfer of S1Altantic salmon smolts.

FIGS. 23A and B are graphical representations of serum calcium,magnesium and sodium levels (mM) over time from Atlantic Salmon S1 APSProcess I treated fish. Each value displays the mean+/−S.D. of a minimumof 10 independent determination from this single representativeexperiment.

FIG. 24 is a graphical representation illustrating the weight (gm) andlength (cm) of representative APS Process II smolts prior to transfer toseawater. This representative sample (n=100) of APS Process II smoltspossess a wide range of body weights (3.95–23 gram) with an average bodyweight of 11.5 gm. Note that all mortalities (n=10) occurred only in thesmaller fish in the transfer group.

FIG. 25 is a graphical representation illustrating the quantitation ofserum concentrations (mM) of calcium, magnesium and sodium in preadultAtlantic salmon subjected to APS Process II after their transfer toseawater. All values shown are the mean+S.D. of a minimum of 10independent samples from a single representative experiment.

FIGS. 26A–C are an alignment illustrating nucleic acid sequences for thePVCR of Atlantic Salmon (SEQ ID NO.: 1), Char (SEQ ID NO.: 3), ChumSalmon (SEQ ID NO.:7), Coho Salmon (SEQ ID NO.:9), King Salmon (SEQ IDNO.:11), Pink Salmon (SEQ ID NO.:13), Sockeye Salmon (SEQ ID NO.:15) andTrout (SEQ ID NO.:5).

FIGS. 27A–B are an alignment illustrating the open reading frame of thepolypeptide sequences for the PVCR of Atlantic Salmon (SEQ ID NO.: 2),Char (SEQ ID NO.: 4), Chum Salmon (SEQ ID NO.: 8), Coho Salmon (SEQ IDNO.: 10), King Salmon (SEQ ID NO.: 12), Pink Salmon (SEQ. ID NO.: 14),Sockeye Salmon (SEQ ID NO.: 16) and Trout (SEQ ID NO.: 6).

FIGS. 28A–B are a diagram illustrating the nucleic acid sequence ofSKCaR (SEQ ID NO.: 17).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for improving the raising ofpre-adult anadromous fish and/or methods for preparing pre-adultanadromous fish for transfer from freshwater to seawater. The methodsinvolve increasing expression and/or altering the sensitivity of aPolyvalent Cation Sensing Receptor (PVCR) (e.g., at least one PVCR). Theinvention relates to increasing expression of the PVCR that affects thefish's ability to adapt to seawater (e.g., to smolt), to undergosmoltification, to survive, to increase growth, to increase foodconsumption, and/or to be less susceptible to disease.

In particular, the methods of the present invention include adding atleast one PVCR modulator to the freshwater, and adding a specially madeor modified feed to the freshwater for consumption by the fish. The feedcontains a sufficient amount of sodium chloride (NaCl) (e.g., betweenabout 1% and about 10% by weight, or about 10,000 mg/kg to about 100,000mg/kg) to significantly increase levels of the PVCR modulator in theserum. This amount of NaCl in the feed causes or induces the pre-adultanadromous fish to drink more freshwater. Since the freshwater containsa PVCR modulator and the fish ingest increased amounts of it, the serumlevel of the PVCR modulator significantly increases in the fish, andcauses increased PVCR expression and/or altered PVCR sensitivity. Thisprocess allows the pre-adult anadromous fish to be “pre-conditioned” andbetter adapt to seawater.

The methods of the present invention pertain to adapting pre-adultanadromous fish to seawater. Anadromous fish are fish that swim fromseawater to freshwater to breed. Anadromous fish include, for example,salmon (e.g, Atlantic Salmon (Salmo salar), Coho Salmon (Oncorhynchuskisutch), Chinook Salmon (Oncorhynchus tshawytscha), Chum Salmon(Oncorhynchus keta), Pink Salmon (Oncorhynchus gorbuscha)), char (e.g.,Arctic Char (Salveninus alpinus)) and trout (e.g., Rainbow Trout(Oncorhynchus mykiss)). Anadromous fish also include fish that areunable to swim to seawater (e.g., landlocked), but have thephysiological mechanisms to adapt to seawater. The term “pre-adultanadromous fish,” as used herein, refers to anadromous fish that havenot yet adapted to seawater. These fish are generally juvenile fish.Pre-adult anadromous fish include, but are not limited to fish that arefingerlings, parr or smolts. As used herein, a “smolt” is a fishundergoing physiological changes that allows the fish to adapt toseawater, or survive upon subsequent transfer to seawater. The term,“smolt,” also refers to a fish that is not at the precise developmentalstage to survive uninjured upon transfer to seawater, but rather is oneof a population of fish wherein, based on a statistical sampling andevaluation, the population of fish is determined to be at aphysiological stage ready for transfer to seawater.

The present invention includes methods for preparing pre-adultanadromous fish undergoing the process of smoltification for transfer toseawater. Smoltification is the stage at which a fish undergoes theacclimation or adaptation from freshwater to seawater. Smoltificationalso refers to a process occurring in pre-adult anadromous fish that isphysiological pre-adaption to seawater while still in freshwater. Thesmolification process varies from species to species. Different speciesof anadromous fish can undergo smoltification at different sizes,weights, and times in the life of the fish. The present inventioninduces the vast majority or all of the pre-adult anadromous fish toundergo this process and be prepared for transfer to seawater.

The pre-adult anadromous fish are maintained in freshwater prior toadding the PVCR modulator. The term, “freshwater,” means water thatcomes from, for example, a stream, river, ponds, public water supply, orfrom other non-marine sources having, for example, the following ioniccomposition: less than about 2 mM of magnesium, calcium and NaCl. Theterm “freshwater” also refers to freshwater to which at least one PVCRmodulator has been added, as described herein.

The PVCR modulator is added to the freshwater in sufficient amounts toincrease expression or alter the sensitivity of the PVCR. A PVCR hasbeen isolated from various tissue of several types of anadromous fishusing molecular biology techniques, as described in Example 9. DNA wasisolated from muscle samples from various species of anadromous fishincluding Atlantic Salmon, Char, Chum Salmon, Coho Salmon, King orChinook Salmon, Pink Salmon, Sockeye Salmon and Trout. The DNA wasamplified using polymerase Chain Reaction (PCR) methodology. Theamplified DNA was purified and subcloned into vectors, and theirsequences were determined, as described in Example 9.

The PVCR, which is located in various tissues (e.g., gill, skin,intestine, kidney, urinary bladder, brain or muscle) of the pre-adultanadromous fish, senses alterations in PVCR modulators including variousions (e.g., divalent cations), for example, in the surrounding water, intheir serum or in the luminal contents of tubules inside the body, suchas kidney, urinary bladder, or intestine. Its ability to sense thesemodulators increases expression of the PVCR, thereby allowing the fishto better adapt to seawater. Increased expression of the PVCR can occur,for example, in one or all tissues.

A “PVCR modulator” is defined herein to mean a compound which increasesexpression of the PVCR, or increases the sensitivity or responsivenessof the PVCR. Such compounds include, but are not limited to, PVCRagonists (e.g., inorganic polycations, organic polycations and aminoacids), Type II calcimimetics, and compounds that indirectly alter PVCRexpression (e.g., 1,25 dihydroxyvitamin D in concentrations of about3,000–10,000 International Units/kg feed), cytokines such as InterleukinBeta, and Macrophage Chemotatic Peptide-1 (MCP-1)). Examples of Type IIcalcimimetics, which increase expression and/or sensitivity of the PVCR,are, for example, NPS-R-467 and NPS-R-568 from NPS Pharmaceutical Inc.,(Salt Lake, Utah, U.S. Pat. Nos. 5,962,314; 5,763,569; 5,858,684;5,981,599; 6,001,884) which can be administered in concentrations ofbetween about 0.1 μM and about 100 μM feed or water. See Nemeth, E. F.et al., PNAS 95: 4040–4045 (1998). Examples of inorganic polycations aredivalent cations including calcium at a concentration between about 2.0and about 10.0 mM and magnesium at a concentration between about 0.5 andabout 10.0 mM; and trivalent cations including, but not limited to,gadolinium (Gd3+) at a concentration between about 1 and about 500 μM.Organic polycations include, but not limited to, aminoglycosides such asneomycin or gentamicin in concentrations of between about 1 and about 8gm/kg feed as well as organic polycations including polyamines (e.g.,polyarginine, polylysine, polyhistidine, polyornithine, spermine,cadaverine, putricine, copolymers of poly arginine/histidine, polylysine/arginine in concentrations of between about 10 μM and 10 mMfeed). See Brown, E. M. et al., Endocrinology 128: 3047–3054 (1991);Quinn, S. J. et al., Am. J. Physiol. 273: C1315–1323 (1997).Additionally, PVCR agonists include amino acids such as L-TryptophanL-Tyrosine, L-Phenylalanine, L-Alanine, L-Serine, L-Arginine,L-Histidine, L-Leucine, L-Isoleucine, and L-Cystine at concentrations ofbetween about 1 and about 10 gm/kg feed. See Conigrave, A. D., et al.,PNAS 97: 4814–4819 (2000). The molar concentrations refer to free orionized concentrations of the PVCR modulator in the freshwater, and doesnot include amounts of bound PVCR modulator (e.g., PVCR modulator boundto negatively charged particles including glass, proteins, or plasticsurfaces). Any combination of these modulators can be added to the wateror to the feed (in addition to the NaCl, as described herein), so longas the combination increases expression and/or sensitivity of the PVCR.

The PVCR modulator can be administered to the fish in a number of ways.The invention encompasses administration of the PVCR in any way that issufficient to increase the expression and/or alter the sensitivity ofthe PVCR. In one embodiment, the PVCR modulator is simply added to thefreshwater in various concentrations, as described herein. A freshwaterenvironment having at least one PVCR modulator is referred to herein asa “PVCR modulator environment.” PVCR modulators that are added to thewater increase expression and/or alter the sensitivity of the PVCR onthe skin and gills of the fish, and can be ingested by the fish, inparticular, when fish are fed feed having between about 1% and about 10%NaCl (e.g., in concentrations between about 1 and about 10 gm/100 gmfeed). In addition to adding NaCl to the feed, the PVCR modulator canalso be added to the feed. Amounts and types of PVCR modulators added tothe feed are also described herein. Other embodiments include subjectingthe fish to the PVCR modulator by “dipping” the fish in the modulator,e.g., organic polycations. The organic polycations can be formulated insuch a way as to allow the polycations to adhere to the skin and gillsof the fish, in sufficient amounts to increase expression of the PVCR.

In one preferred embodiment, the present invention is practiced byadding a combination of two PVCR agonists to the freshwater. Inparticular, calcium and magnesium are added to the freshwater to bringthe concentrations of each to between about 2.0 mM and about 10.0 mM ofcalcium, and between about 0.5 mM and about 10.0 mM of magnesium. Inaddition to adding calcium and magnesium to the water, these ranges ofion concentrations can be achieved by providing a brackish water (e.g.,diluted seawater) environment for the fish.

Calcium and magnesium can come from a variety of sources, that whenadded to the water, the calcium and/or magnesium levels increaseexpression of the PVCR, and/or are within the stated ranges. Sources ofcalcium and magnesium can be a mixture of a variety of compounds, oreach can come from a substantially uniform or pure compound. Sources ofcalcium include, for example, Ca(CO₃)₂, CaCl₂,CaSO₄, and Ca(OH)₂ andsources of magnesium include, for example, MgCl₂, MgSO₄, MgBr₂, andMgCO₃.

In one embodiment, the invention includes intermittent (e.g.,interrupted) as well as continuous (e.g., non-interrupted) exposure tofreshwater having at least one PVCR modulator, while on the NaCl diet.Intermittent exposure to the PVCR can occur so long as the PVCRexpression and/or altered sensitivity remains increased. Continuousmaintenance in or exposure to freshwater having at least one PVCRmodulator is shown in Examples 2 and 7.

The process of the present invention pre-conditions the fish andprepares them for transfer. The pre-adult anadromous fish are maintainedin a freshwater environment having a PVCR modulator long enough toincrease the expression and/or alter sensitivity of the PVCR. The lengthof time depends on the physiological and physical maturity of the fish.Some fish will more readily adapt to the environment, and increase theirexpression and/or alter the sensitivity of their PVCR, while others willneed more time to do so. Factors that can influence the length of timenecessary to increase the expression and/or alter sensitivity of thePVCR include, but are not limited to, size of the fish, level of PVCRexpression or sensitivity, if any, prior to addition of the PVCRmodulator to the freshwater, the fish's ability to excrete the PVCRmodulator and ions, the fish's surface to volume ratio, etc. Therefore,the length of time the fish is maintained can range from about 7 days toseveral months (e.g., 7, 14, 21, 30, 45, 90 and 120 days). The fish canalso be maintained indefinitely so long as the fish are maintained infreshwater having the PVCR modulator and being fed a NaCl diet. Forexample, salmon, trout or char weighing less than 10 gms can bemaintained in freshwater having a PVCR modulator, and fed a NaCl dietfor at least about 180 days, prior to transfer to seawater.

The invention further includes adding feed to the freshwater. Thefrequency and amounts of feed that fish are fed, are taught in the art.Generally, the fish are fed 1–3 times a day, totaling about 0.25–5.0%body weight/day. The feed has enough NaCl to contribute to a significantincreased level of the PVCR modulator in the serum of the pre-adultanadromous fish. More specifically, NaCl has at least two effects. Thefirst occurs when sufficient amounts of NaCl is present in the feed. Thepresence of NaCl in the feed causes the pre-adult anadromous fish todrink more water from the surrounding environment. Second, NaCl is adirect negative PVCR modulator, and works to decreases PVCR sensitivity.Despite NaCl's effect in decreasing sensitivity, it surprisinglyincreases PVCR expression when fish are fed a NaCl diet and thesurrounding freshwater environment has at least one PVCR modulator itin. The increase in the ingestion of freshwater having PVCR modulatorscauses an overall increase of the serum levels of PVCR modulators.

The present invention also relates to an aquatic food composition. Thefeed contains between about 1%–10% of NaCl by weight, or between about10,000 mg of NaCl/kg of feed and about 100,000 mg of NaCl/kg of feed(e.g., 12,000 mg/kg). The feed is referred to herein as a “NaCl diet.”The NaCl can be combined with other sodium salts to confer the desiredeffect of increasing PVCR expression, altering PVCR sensitivity and/orinducing the fish to drink more. Hence, as used herein, the term NaCl,includes a substantially pure compound, and mixtures of NaCl with othersources of sodium. The feed can further include a PVCR modulator, and inparticular a PVCR agonist such as an amino acid. In one embodiment, thefeed has between about 1% and about 10% NaCl by weight and an amino acidsuch as tryptophan in an amount between about 1 and about 10 gm/kg.

The feed can be made in a number of ways, so long as the properconcentration of NaCl is present. The feed can be made, for example, byreformulating the feed, or by allowing the feed to absorb a solutionhaving the NaCl and optionally, adding a PVCR modulator. A top dressingcan be added for palatability. Example 8 describes in detail one way tomake the feed.

Another embodiment of the present invention includes feeding pre-adultanadromous fish feed having between 1% and 10% NaCl by weight when thefish are maintained in a freshwater environment having between about 2.0and about 10.0 mM of calcium, and between about 0.5 mM and about 10.0 mMof magnesium. When this embodiment of the present invention is carriedout, the levels of calcium, magnesium and/or sodium in the serum of thepre-adult anadromous fish increases, as compared to identically pairedfish maintained in freshwater, between about 1% and 60%, between about1% and 40%, and between about 1% and 15%, respectively.

In another embodiment, the fish, while in the freshwater having the PVCRmodulator, are also exposed to a photoperiod. A photoperiod refers toexposing the fish to light (e.g., sunlight, incandescent light orfluorescent light). Preferably, the photoperiod is substantiallycontinuous, or occurs long enough to increase growth and/or inducesmotification. The photoperiod can occur for at least about 12 hourswithin a 24 hour interval, or for longer periods such as about 14, 16,18, 20, 22 or preferably, about 24 hours.

After being exposed to the steps of the present invention, the pre-adultanadromous fish are transferred to seawater. The term, “seawater,” meanswater that comes from the sea, or water which has been formulated tosimulate the chemical and mineral composition of water from the sea. Themajor elemental composition of the prepared seawater preferably fallssubstantially within the range of the major elemental composition of thenatural seawater (e.g., having the following ionic composition: greaterthan 30 mM of magnesium, greater than about 6 mM of calcium, and greaterthan about 300 mM NaCl). Methods of preparing artificial seawater areknown in the art and are described in, for instance, U.S. Pat. No.5,351,651.

When performing the methods of the present invention on pre-adultanadromous fish, the fish exhibit significant increased growth and foodconsumption, as compared to pre-adult anadromous fish that are notsubjected to the present invention. Upon transfer to seawater, fish thatare not subjected to the steps of the present invention generallyexperience osmotic stress, reduced or no food consumption, and evendeath. Osmotic stress results from differences in the osmotic pressurebetween the surrounding environment and body compartments of the fish.This disturbs the homeostatic equilibrium of the fish and results indecreased growth, reproductive failure and reduced resistance todisease. The fish that have been preconditioned by the steps of thepresent invention do not experience a significant amount of osmoticstress, and begin feeding on or soon after transfer to seawater. As aresult, the fish also grow earlier. In particular, pre-adult anadromousfish that ingested a feed having between about 1% and about 10% NaCl,and between about 1 gm and about 10 gms per kg of feed of an amino acid,exhibit a substantial increase in growth. In the experiments, the fishadapted by the present invention have shown as much as about 65%increased growth during the same interval of time, as compared toidentically paired fish that did not undergo the steps of the presentinvention and were transferred to seawater. See Table 4 of Example 2.Elimination of low feeding or poorly feeding osmotically stressed fishin a group improves the overall feed conversion ratio of the entiregroup. Optimal feeding and growth after seawater transfer by all membersof the group of pre-conditioned fish will permit better feed utilizationand improve the overall yield of production when fish reach market size.

Similarly, the present invention allows for decreasing or reducing thetime between generations of pre-adult anadromous fish. These fish beginbreeding earlier because the present invention increases their growth,as described herein. Since 2–3 years are required to obtain sexuallymature fish, attempts to engage in selective breeding of traits requiresthis 2–3 year interval before a given trait can be selected for and thefish exhibit that trait breed. Improvements in growth and time to reachmaturity produced by the invention reduce the time interval required toreach sexual maturity in fish by as much as about 6 months to about 12months. Reducing the interval for breeding allows for the production ofmore fish, and the improved selection of fish that possess traits otherthan those that are better able to adapt to seawater (e.g., select forfish that have improved taste, increased filet thickness, increased α3omega fatty acid content, or fish that are more readily able to increasePVCR expression).

Prior to the present invention, anadromous fish that are transferredfrom freshwater to seawater are generally transferred at a particularsize, referred to as “critical size.” The critical size varies fromspecies to species, but generally refers to a minimum size at which afish can be transferred to seawater. The critical size for salmon, troutand char is between about 50 and about 100 gms, between about 70 andabout 120 gms, and greater than 100 gms, respectively. Critical sizesfor Coho, King, and Sockeye Salmon are between about 10 and about 15gms, between about 20 and 40 gms and between about 1 and about 2 gms,respectively. Chum and Pink Salmon each have a critical size about lessthan 3 gms.

Prior to the invention, a population of pre-adult anadromous fish havingattained a mean critical size were transferred to seawater. Some of thefish are physiologically ready for the transfer, while others are not.This is one of the reasons for the increased mortality rate upontransfer to seawater. The methods of the present inventionphysiologically prepares all or mostly all of the fish for transfer toseawater by increasing PVCR expression and/or sensitivity, and/or byinducing smoltification. Greater than about 80% (e.g., 90%, 95%, 100%)undergo smoltification and are ready for transfer to seawater. In fact,in one experiment, when performing the steps of the present invention onAtlantic Salmon (e.g., subjecting the fish to a PVCR modulatorenvironment and a NaCl diet), close to 100% of the Atlantic Salmonunderwent smoltification. See Example 2. Hence, the methods of thepresent invention include methods of preparing pre-adult anadromous fishfor transfer to seawater, as well as inducing smotification in pre-adultanadromous fish.

Since the methods of the present invention increase the expressionand/or sensitivity of the PVCR in pre-adult anadromous fish, theysurvive better when transferred to seawater. The reduced osmotic stressresults in reduced mortality. In one case, certain populations ofpre-adult anadromous fish that did not undergo the methods of thepresent invention exhibit a 100% mortality rate after transfer toseawater (see FIG. 9, Example 2), while other populations of pre-adultanadromous fish that did not undergo the methods of the invention havesurvival rates of only between about 40% and 70%. See Table I, Example2. This occurs because the fish experience osmotic shock whentransferred to seawater which has a very different ionic compositionthan freshwater. However, when preconditioned by the methods of thepresent invention, the fish exhibit a survival rate that issignificantly greater than the rate for unconditioned fish (e.g.,between about 80% about 100%). In fact, when performing the presentinvention on Atlantic Salmon, 99% of the fish survived transfer toseawater after 5 days, as compared to 33% of fish that did not undergothe steps of the present invention in one experiment. See Table I ofExample 2. Hence, the present invention embodies methods of reducing themortality rate after pre-adult anadromous fish are transferred toseawater.

Not only is the present invention useful in reducing mortality ratesafter transfer to seawater, the present invention is also used toincrease survival rates in freshwater prior to transfer. Prior to thediscovery of the present invention, a “smolt window” existed in whichthe hatcheries transferred the pre-adult anadromous fish to seawater, orelse the fish will die if they continue to remain in freshwater afterthey undergo smoltification. The PVCR modulator environment and the NaCldiet of the present invention allow the fish to continue to thriveindefinitely. The fish continue to consume feed and grow. When thepresent invention was performed on Atlantic Salmon, 99% of the fishsurvived and thrived for at least 45 days in freshwater. In contrast,only 67% of the fish that did not undergo the steps of the inventionsurvived after 45 days in freshwater in one experiment. See Example 2.

The present invention also includes methods for transferring to seawaterpre-adult anadromous fish having smaller weights, as compared to theindustry recognized critical size for the particular species of fish.The methods of the present invention, as described herein, increase PVCRexpression in fish that are smaller than those normally transferred toseawater, or those undergoing or about to undergo smoltification. Thesemethods include transferring a parr, the stage of a juvenile fish priorto becoming a smolt, to seawater. Parr is a life stage of pre-adultanadromous fish that occurs after maturation of alevins or yolk sac fry.Parr or fingerlings display characteristic ovid stripes or parr marksalong their flanks, and normally undergo growth and development infreshwater prior to smoltification. The term “parr” is a term that isknown in the art. As yolk sac fry continue to feed, they grow intolarger parr. Parr can possess a wide range of body weights depending onconditions under which they are grown. The weights of parr vary fromspecies to species. Body weights for parr vary significantly with arange from about 0.5 gms to about 70 gms. Carrying out the presentinvention in one experiment, as described herein, results in a transferof Atlantic Salmon parr weighing as little as between about 13% andabout 18.5% of the critical size weight (between about 70 and about 100gms), or about 13 gms Adding a PVCR modulator to the feed (e.g., anamino acid such as a tryptophan), in addition to the NaCl diet, allowsseawater transfer of fish having particularly low weights. See Example2.

The present invention additionally provides methods for transferringpre-adult anadromous fish into seawater having warmer temperatures(e.g., 14° C. and 19° C.), as compared to water temperatures into whichthese fish have been transferred in the past. Since the fish experiencereduced or little osmotic stress when transferred to seawater using themethods of the present invention, the fish are able to withstandtransfer into higher water temperatures without exhibiting an increasein mortality rates. See Example 2.

The methods of the present invention also decrease the incidence ofdisease among the smolts and the growing salmon. Because smolts treatedwith the methods of the present invention experience less stress upontransfer to seawater, their immune functions are stronger, and they areless susceptible to parasitic, viral, bacterial and fungal diseases.Fish not treated with the methods described herein are more susceptibleto such diseases, and can serve as reservoirs of disease, capable ofinfecting healthy fish.

Methods of Assessment of the PVCR

The present invention includes methods of detecting the level of thePVCR to determine whether fish are ready for transfer from freshwater toseawater. Methods that measure PVCR levels include several suitableassays. Suitable assays encompass immunological methods, such as FACSanalysis, radioimmunoassay, flow cytometry, enzyme-linked immunosorbentassays (ELISA) and chemiluminescence assays. Any method known now ordeveloped later can be used for measuring PVCR expression.

Antibodies reactive with the PVCR or portions thereof can be used. In apreferred embodiment, the antibodies specifically bind with the PVCR ora portion thereof. The antibodies can be polyclonal or monoclonal, andthe term antibody is intended to encompass polyclonal and monoclonalantibodies, and functional fragments thereof. The terms polyclonal andmonoclonal refer to the degree of homogeneity of an antibodypreparation, and are not intended to be limited to particular methods ofproduction.

In several of the preferred embodiments, immunological techniques detectPVCR levels by means of an anti-PVCR antibody (i.e., one or moreantibodies). The term “anti-PVCR” antibody includes monoclonal and/orpolyclonal antibodies, and mixtures thereof.

Anti-PVCR antibodies can be raised against appropriate immunogens, suchas isolated and/or recombinant PVCR or portion thereof (includingsynthetic molecules, such as synthetic peptides). In one embodiment,antibodies are raised against an isolated and/or recombinant PVCR orportion thereof (e.g., a peptide) or against a host cell which expressesrecombinant PVC R. In addition, cells expressing recombinant PVCR, suchas transfected cells, can be used as immunogens or in a screen forantibody which binds receptor.

Any suitable technique can prepare the immunizing antigen and producepolyclonal or monoclonal antibodies. The art contains a variety of thesemethods (see e.g., Kohler et al., Nature, 256: 495–497 (1975) and Eur.J. Immunol. 6: 511–519 (1976); Milstein et al., Nature 266: 550–552(1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D.Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y.); Current Protocols In MolecularBiology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al.,Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991)).Generally, fusing a suitable immortal or myeloma cell line, such asSP2/0, with antibody producing cells can produce a hybridoma. Animalsimmunized with the antigen of interest provide the antibody producingcell, preferably cells from the spleen or lymph nodes. Selective cultureconditions isolate antibody producing hybridoma cells while limitingdilution techniques produce them. Researchers can use suitable assayssuch as ELISA to select antibody producing cells with the desiredspecificity.

Other suitable methods can produce or isolate antibodies of therequisite specificity. Examples of other methods include selectingrecombinant antibody from a library or relying upon immunization oftransgenic animals such as mice.

According to the method, an assay can determine the level of PVCR in abiological sample. In determining the amounts of PVCR, an assay includescombining the sample to be tested with an antibody having specificityfor the PVCR, under conditions suitable for formation of a complexbetween antibody and the PVCR, and detecting or measuring (directly orindirectly) the formation of a complex. The sample can be obtaineddirectly or indirectly, and can be prepared by a method suitable for theparticular sample and assay format selected.

In particular, tissue samples, e.g., gill tissue samples, can be takenfrom fish after they are anaesthetized with MS-222. The tissue samplesare fixed by immersion in 2% paraformaldehyde in appropriate Ringerssolution corresponding to the osmolality of the fish, washed in Ringers,then frozen in an embedding compound, e.g., O.C.T.™ (Miles, Inc.,Elkahart, Ind., USA) using methylbutane cooled with liquid nitrogen.After cutting 8–10 μ tissue sections with a cryostat, individualsections are subjected to various staining protocols. For example,sections are: 1) blocked with goat serum or serum obtained from the samespecies of fish, 2) incubated with rabbit anti-CaR or anti-PVCRantiserum, and 3) washed and incubated with peroxidase-conjugatedaffinity-purified goat antirabbit antiserum. The locations of the boundperoxidase-conjugated goat antirabbit antiserum are then visualized bydevelopment of a rose-colored aminoethylcarbazole reaction product.Individual sections are mounted, viewed and photographed by standardlight microscopy techniques. The anti-CaR antiserum used to detect fishPVCR protein is raised in rabbits using a 23-mer peptide correspondingto amino acids numbers 214–236 localized in the extracellular domain ofthe RaKCaR protein. The sequence of the 23-mer peptide is:ADDDYGRPGIEKFREEAEERDIC (SEQ ID NO.: 19) A small peptide with thesequence DDYGRPGIEKFREEAEERDICI (SEQ ID NO.: 20) or ARSRNSADGRSGDDLPC(SEQ ID NO.: 21) can also be used to make antisera containing antibodiesto PVCRs. Such antibodies can be monoclonal, polyclonal or chimeric.

Suitable labels can be detected directly, such as radioactive,fluorescent or chemiluminescent labels. They can also be indirectlydetected using labels such as enzyme labels and other antigenic orspecific binding partners like biotin. Examples of such labels includefluorescent labels such as fluorescein, rhodamine, chemiluminescentlabels such as luciferase, radioisotope labels such as 32P, 125I, 131I,enzyme labels such as horseradish peroxidase, and alkaline phosphatase,β-galactosidase, biotin, avidin, spin labels and the like. The detectionof antibodies in a complex can also be done immunologically with asecond antibody which is then detected (e.g., by means of a label).Conventional methods or other suitable methods can directly orindirectly label an antibody.

In performing the method, the levels of the PVCR that are distinct fromthe control. Icreased levels or the presence of PVCR expression, ascompared to a control, indicate that the fish or the population of fishfrom which a statistically significant amount of fish were tested, areready for transfer to freshwater. A control refers to a level of PVCR,if any, from a fish that is not subjected to the steps of the presentinvention, e.g., not subjected to freshwater having a PVCR modulatorand/or not fed a NaCl diet. For example, FIGS. 13 and 18 show that fishnot subjected to the present invention had no detectable PVCR level,whereas fish that were subjected to the steps of the invention had PVCRlevels that were easily detected.

The PVCRs can also be assayed by Northern blot analysis of mRNA fromtissue samples. Northern blot analysis from various shark tissues hasrevealed that the highest degree of PVCRs expression is in gill tissue,followed by the kidney and the rectal gland. There appear to be at leastthree distinct mRNA species of about 7 kb, 4.2 kb and 2.6 kb.

The PVCRs can also be assayed by hybridization, e.g., by hybridizing oneof the PVCR sequences provided herein (e.g., SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15) or an oligonucleotide derived from one of the sequences, toa DNA-containing tissue sample from a fish. Such a hybridizationsequence can have a detectable label, e.g., radioactive, fluorescent,etc., attached, to allow to detection of hybridization product. Methodsfor hybridization are well known, and such methods are provided in U.S.Pat. No. 5,837,490, by Jacobs et al., the entire teachings of which areherein incorporated by reference in their entirety. The design of theoligonucleotide probe should preferably follow these parameters: (a) itshould be designed to an area of the sequence which has the fewestambiguous bases (“N's”), if any, and (b) it should be designed to have aT_(m) of approx. 80° C. (assuming 2° C. for each A or T and 4 degreesfor each G or C).

Stringency conditions for hybridization refers to conditions oftemperature and buffer composition which permit hybridization of a firstnucleic acid sequence to a second nucleic acid sequence, wherein theconditions determine the degree of identity between those sequenceswhich hybridize to each other. Therefore, “high stringency conditions”are those conditions wherein only nucleic acid sequences which are verysimilar to each other will hybridize. The sequences can be less similarto each other if they hybridize under moderate stringency conditions.Still less similarity is needed for two sequences to hybridize under lowstringency conditions. By varying the hybridization conditions from astringency level at which no hybridization occurs, to a level at whichhybridization is first observed, conditions can be determined at which agiven sequence will hybridize to those sequences that are most similarto it. The precise conditions determining the stringency of a particularhybridization include not only the ionic strength, temperature, and theconcentration of destabilizing agents such as formamide, but also onfactors such as the length of the nucleic acid sequences, their basecomposition, the percent of mismatched base pairs between the twosequences, and the frequency of occurrence of subsets of the sequences(e.g., small stretches of repeats) within other non-identical sequences.Washing is the step in which conditions are set so as to determine aminimum level of similarity between the sequences hybridizing with eachother. Generally, from the lowest temperature at which only homologoushybridization occurs, a 1% mismatch between two sequences results in a1° C. decrease in the melting temperature (T_(m)) for any chosen SSCconcentration. Generally, a doubling of the concentration of SSC resultsin an increase in the T_(m) of about 17° C. Using these guidelines, thewashing temperature can be determined empirically, depending on thelevel of mismatch sought. Hybridization and wash conditions areexplained in Current Protocols in Molecular Biology (Ausubel, F. M. etal., eds., John Wiley & Sons, Inc., 1995, with supplemental updates) onpages 2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.

High stringency conditions can employ hybridization at either (1) 1×SSC(10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to 7.0 with 1M HCl), 1% SDS (sodium dodecyl sulfate), 0.1–2 mg/ml denatured calfthymus DNA at 65° C., (2) 1×SSC, 50% formamide, 1% SDS, 0.1–2 mg/mldenatured calf thymus DNA at 42° C., (3) 1% bovine serum albumen(fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄=134 gNa₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1–2 mg/ml denaturedcalf thymus DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH7.6), 1× Denhardt's solution (100×=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1–2 mg/ml denatured calf thymusDNA at 42° C., (5) 5×SSC, 5× Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 65° C., or (6) 5×SSC, 5× Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at42° C., with high stringency washes of either (1) 0.3–0.1×SSC, 0.1% SDSat 65° C., or (2) 1 mM Na₂EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS at 65° C.The above conditions are intended to be used for DNA—DNA hybrids of 50base pairs or longer. Where the hybrid is believed to be less than 18base pairs in length, the hybridization and wash temperatures should be5–10° C. below that of the calculated T_(m) of the hybrid, where T_(m)in ° C.=(2×the number of A and T bases)+(4×the number of G and C bases).For hybrids believed to be about 18 to about 49 base pairs in length,the T_(m) in ° C.=(81.5° C.+16.6(log₁₀M)+0.41(% G+C)−0.61 (%formamide)−500/L), where “M” is the molarity of monovalent cations(e.g., Na⁺), and “L” is the length of the hybrid in base pairs.

Moderate stringency conditions can employ hybridization at either (1)4×SSC, (10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to 7.0with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1–2 mg/ml denaturedcalf thymus DNA at 65° C., (2) 4×SSC, 50% formamide, 1% SDS, 0.1–2 mg/mldenatured calf thymus DNA at 42° C., (3) 1% bovine serum albumen(fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄=134 gNa₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1–2 mg/ml denaturedcalf thymus DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH7.6), 1× Denhardt's solution (100×=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1–2 mg/ml denatured calf thymusDNA at 42° C., (5) 5×SSC, 5× Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 65° C., or (6) 5×SSC, 5× Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at42° C., with moderate stringency washes of 1×SSC, 0.1% SDS at 65° C. Theabove conditions are intended to be used for DNA—DNA hybrids of 50 basepairs or longer. Where the hybrid is believed to be less than 18 basepairs in length, the hybridization and wash temperatures should be 5–10°C. below that of the calculated T_(m) of the hybrid, where T_(m) in °C.=(2×the number of A and T bases)+(4×the number of G and C bases). Forhybrids believed to be about 18 to about 49 base pairs in length, theT_(m) in ° C.=(81.5° C.+16.6(log₁₀M)+0.41(% G+C)−0.61 (%formamide)−500/L), where “M” is the molarity of monovalent cations(e.g., Na⁺), and “L” is the length of the hybrid in base pairs.

Low stringency.conditions can employ hybridization at either (1) 4×SSC,(10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to 7.0 with 1M HCl), 1% SDS (sodium dodecyl sulfate), 0.1–2 mg/ml denatured calfthymus DNA at 50° C., (2) 6×SSC, 50% formamide, 1% SDS, 0.1–2 mg/mldenatured calf thymus DNA at 40° C., (3) 1% bovine serum albumen(fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄=134 gNa₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1–2 mg/ml denaturedcalf thymus DNA at 50° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH7.6), 1× Denhardt's solution (100×=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1–2 mg/ml denatured calf thymusDNA at 40° C., (5) 5×SSC, 5× Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 50° C., or (6) 5×SSC, 5× Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at40° C., with low stringency washes of either 2×SSC, 0.1% SDS at 50° C.,or (2) 0.5% bovine serum albumin (fraction V), 1 mM Na₂EDTA, 40 mMNaHPO₄ (pH 7.2), 5% SDS. The above conditions are intended to be usedfor DNA—DNA hybrids of 50 base pairs or longer. Where the hybrid isbelieved to be less than 18 base pairs in length, the hybridization andwash temperatures should be 5–10° C. below that of the calculated T_(m)of the hybrid, where T_(m) in ° C.=(2×the number of A and Tbases)+(4×the number of G and C bases). For hybrids believed to be about18 to about 49 base pairs in length, the T_(m) in ° C.=(81.5°C.+16.6(log₁₀M)+0.41(% G+C)−0.61 (% formamide)−500/L), where “M” is themolarity of monovalent cations (e.g., Na⁺), and “L” is the length of thehybrid in base pairs.

Hence, the present invention includes kits for the detection of the PVCRor the quantification of the PVCR having either antibodies specific forthe PVCR or a portion thereof, or a nucleic acid sequence that canhybridize to the nucleic acid of the PVCR.

Alterations in the expression or sensitivity of PVCRs could also beaccomplished by introduction of a suitable transgene. Suitabletransgenes would include either the PVCR gene itself or modifier genesthat would directly or indirectly influence PVCR gene expression.Methods for successful introduction, selection and expression of thetransgene in fish oocytes, embryos and adults are described in Chen, T Tet al., Transgenic Fish, Trends in Biotechnology 8:209–215 (1990).

The present invention is further and more specifically illustrated bythe following Examples, which are not intended to be limiting in anyway.

EXEMPLIFICATION Example 1 Polyvalent Cation-Sensing Receptors (PVCRs)Serve as Salinity Sensors in Fish

Polyvalent cation-sensing receptors (PVCRS) serve as salinity sensors infish. These receptors are localized to the apical membranes of variouscells within the fish's body (e.g., in the gills, intestine, kidney)that are known to be responsible for osmoregulation. A full-lengthcation receptor (CaR) from the dogfish shark has been expressed in humanHEK cells. This receptor was shown to respond to alterations in ioniccompositions of NaCl, Ca2+ and Mg2+ in extracellular fluid bathing theHEK cells. The ionic concentrations responded to encompassed the rangewhich includes the transition from freshwater to seawater. Expression ofPVCR mRNA is also increased in fish after their transfer from freshwaterto seawater, and is modulated by PVCR agonists. Partial genomic clonesof PVCRs have also been isolated from other fish species, includingwinter and summer flounder and lumpfish, by using nucleic acidamplification with degenerate primers.

This method was also used to isolate partial genomic clones of PVCRs forAtlantic salmon (FIG. 1), arctic char (FIG. 2) and rainbow trout (FIG.3). The degenerate oligonucleotide primers used were 5′-TGT CKT GGA CGGAGC CCT TYG GRA TCG C-3′ (SEQ ID NO:22) AND 5′-GGC KGG RAT GAA RGA KATCCA RAC RAT GAA G-3′ (SEQ ID NO:23), where K is T or G, Y is C or T, andR is A or G. The degenerate oligos were generated by standardmethodologies (Preston, G. M., 1993, “Polymerase chain reaction withdegenerate oligonucleotide primers to clone gene family members,” in:Methods in Mol. Biol., vol. 58, ed. A. Harwood, Humana Press, pp.303–312). Genomic bands from these three species were amplified,purified by agarose gel electrophoresis, ligated into an appropriateplasmid vector (salmon and arctic char species-pT7 Blue (Novagen,Madison, Wis.; trout used pGem-T (Promega Biotech. Madison, Wis.), andtransformed into an appropriate bacterial host strain salmon and arcticchar-pT7 vector with NovaBlue (Novagen, Madison, Wis.) and trout pGEM-Tused JM-109 E. coli cell which was then grown in liquid medium. Theplasmids and inserts were purified from the host cells, and sequenced.FIG. 4 shows the deduced amino acid sequences and alignment for thePVCRs from Atlantic salmon, arctic char and rainbow trout, relative tothe PVCR from the kidney of the dogfish shark (Squalus acanthias).

Example 2 Survival and Growth of Pre-Adult Anadromous Fish Using theMethods of the Present Invention

An important feature of current salmon farming is the placement of smoltfrom freshwater hatcheries to ocean netpens. Present day methods usesmolt that have attained a critical size of approximately 70–110 gramsbody weight. The present invention can either be utilized both toimprove the ocean netpen transfer of standard 70–110 grams smolt as wellas permit the successful ocean netpen transfer of smolt weighing only 30grams. For standard 70–100 gram smolt, application of the inventioneliminates the phenomenon known as “smolt window” and permits fish to bemaintained and transferred into ocean water at 15° C. or higher. Use ofthe invention in 30 gram or smaller smolt permits greater utilization offreshwater hatchery capacities followed by successful seawater transferto ocean netpens. In both cases, fish that undergo the steps of theinvention feed vigorously within a short interval of time after transferto ocean netpens and thus exhibit rapid growth rates upon transfer toseawater.

FIG. 5 shows in schematic form the key features of current aquacultureof Atlantic salmon in ocean temperatures present in Europe and Chile.Eggs are hatched in inland freshwater hatcheries and the resulting frygrow into fingerlings and parr. Faster growing parr are able to undergosmoltification and placement in ocean netpens as S0 smolt (70 gram)during year 01. In contrast, slower growing parr are smoltified in year02 and placed in netpens as S1 smolt (100 gram). In both S0 and S1transfers to seawater, the presence of cooler ocean and freshwatertemperatures are desired to minimize the stress of osmotic shock tonewly transferred smolt. This is particularly true for S1 smolt sincefreshwater hatcheries are often located at significant distances fromocean netpen growout sites and their water temperatures rise rapidlyduring early summer. Thus, the combination of rising water temperaturesand the tendency of smolt to revert or die when held for prolongedintervals in freshwater produces a need to transfer smolt into seawaterduring the smolt window.

Standard smolts that are newly placed in ocean netpens are not able togrow optimally during their first 40–60 day interval in seawater becauseof the presence of osmotic stress that delays their feeding. Thisinterval of osmotic adaptation prevents the smolts from taking advantageof the large number of degree days present immediately after eitherspring or fall placement. The combination of the presence of the smoltwindow together with delays in achieving optimal smolt growth prolongthe growout interval to obtain market size fish. This is particularlyproblematic for S0's since the timing of their harvest is complicated bythe occurrence of grilsing in maturing fish that are exposed toreductions in ambient photoperiod.

Methods

The following examples refer to APS Process I and APS Process IIthroughout. APS stands for “AquaBio Products Sciences®, L.L.C.” APSProcess I is also referred to herein as “SUPERSMOLT™ I Process” or“Process I.” An “APS Process I” fish or smolt refers to a fish or smoltthat has undergone the steps of APS Process I. An APS Process I smolt isalso referred to as a “SUPERSMOLT™ I” or a “Process I” smolt. Likewise,APS Process II is also referred to herein as “SUPERSMOLT™ II Process” or“Process II.” An “APS Process II” fish or smolt refers to a fish orsmolt that has undergone the steps of APS Process II. An APS Process IIsmolt is also referred to as a “SUPERSMOLT™ II” or a “Process II” smolt.

APS Process I: Pre-adult anadromous fish (this includes bothcommercially produced S0, S1 or S2 smolts as well as smaller parr/smoltfish) are exposed to or maintained freshwater containing either 2.0–10.0mM Calcium and 0.5–10.0 mM Magnesium ions. This water is prepared byaddition of calcium carbonate and/or chloride and magnesium chloride tothe freshwater. Fish are fed with feed pellets containing 7%(weight/weight) NaCl. See Example 8 for further details regarding thefeed. Fish are exposed to or maintained in this regimen of water mixtureand feed for a total of 30–45 days, using standard hatchery caretechniques. Water temperatures vary between 10–16° C. Fish are exposedto a constant photoperiod for the duration of APS Process I. Afluorescent light is used for the photoperiod.

APS Process II: Pre-adult anadromous fish (this includes bothcommercially produced S0, S1 or S2 smolts as well as smaller parr/smoltfish) are exposed to or maintained in freshwater containing 2.0–10.0 mMCalcium and 0.5–10.0 mM Magnesium ions. This water is prepared byaddition of calcium carbonate and/or chloride and magnesium chloride tothe freshwater. Fish are fed with feed pellets containing 7%(weight/weight) NaCl and either 2 gm or 4 gm of L-Tryptophan per kg offeed. See Example 8 for further details regarding the feed. Fish areexposed to or maintained in this regimen of water mixture and feed for atotal of 30–45 days using standard hatchery care techniques. Watertemperatures vary between 10–16° C. Fish are exposed to a constantphotoperiod for the duration of APS Process II. A fluorescent light isused for the photoperiod.

Results and Discussion:

SECTION I: Demonstration of the Benefits of the APS Process I ForAtlantic Salmon, Trout and Arctic Char.

Demonstration of the Benefits of the APS Process I for Atlantic Salmon:

APS Process I increases the survival of small Atlantic Salmon S2 smoltafter their transfer to seawater when compared to matched freshwatercontrols. Optimal survival is achieved by using the complete processconsisting of both the magnesium and calcium water mixture as well asNaCl diet. In contrast, administration of calcium and magnesium eithervia the food only or without NaCl dietary supplementation does notproduce results equivalent to APS Process I.

Table 1 shows data obtained from Atlantic salmon S2 smolts less than 1year old weighing approximately 25 gm. This single group of fish wasapportioned into 4 specific groups as indicated below and each weremaintained under identical laboratory conditions except for thevariables tested. All fish were maintained at a water temperature of9–13° C. and a continuous photoperiod for the duration of theexperiment.

The control freshwater group that remained in freshwater for the initial45 day interval experienced a 33% mortality rate under these conditionssuch that only 67% were able to be transferred to seawater. Aftertransfer to seawater, this group also experienced high mortality whereonly one half of these smolts survived. Inclusion of calcium (10 mM) andmagnesium (5 mM) within the feed offered to smolt (Ca2+/Mg2+diet)reduced survival as compared to controls both in freshwater (51% vs 67%)as well after seawater transfer (1% vs 50%). In contrast, inclusion of10 mM Ca2+ and 5 mM Mg2+ in the freshwater (APS Process I Water Only)improved smolt survival in APS Process I water as well as after transferof smolt to seawater. However, optimal results were obtained (99%survival in both the APS Process I water mixture as well as afterseawater transfer) when smolt were maintained in APS Process I watermixture and fed a diet supplemented with 7% sodium chloride.

TABLE 1 Comparison of the Survival of Atlantic Salmon S2 Smolts AfterVarious Treatments Parameter Control Ca2+/Mg2+ APS Water APS Water +Sampled Freshwater Diet Only NaCl Diet Starting # 66 70 74 130 of fish #of fish 44 36 67 129 % of fish   67%   51%   91%   99% surviving after45 days in freshwater or APS mixture # of fish 22  2 60 128 % of fish  50%   1%   90%   99% surviving 5 days after transfer to seawater¹Survival percentages expressed as rounded whole numbersApplication of the APS Process I to the Placement of 70–100 gm smolts inseawater.

These data show that use of the APS Process I eliminates the “smoltwindow” and provides for immediate smolt feeding and significantimprovement in smolt growth rates.

Experimental Protocol:

Smolts derived from the St. John strain of Atlantic salmon produced bythe Connors Brothers Deblois Hatchery located in Cherryfield, Me., USAwere utilized for this large scale test. Smolts were produced usingstandard practices at this hatchery and were derived from a January 1999egg hatching. All smolts were transferred with standard commerciallyavailable smolt trucks and transfer personnel. S1 smolt were purchasedduring Maine's year 2000 smolt window and smolt deliveries were takenbetween the dates of 29 Apr. 2000–15 May 2000. Smolts were eithertransferred directly to Polar Circle netpens (24 m diameter) located inBlue Hill Bay Maine (Controls) or delivered to the treatment facilitywhere they were treated with APS Process I for a total of 45 days. Afterreceiving the APS Process I treatment, the smolt were then transportedto the identical Blue Hill Bay netpen site and placed in an adjacentrectangular steel cage (15 m×15 m×5 m) for growout. Both groups of fishreceived an identical mixture of moist (38% moisture) and dry (10%moisture) salmonid feed (Connors Bros). Each of the netpens were fed byhand or feed blower to satiation twice per day using cameravisualization of feeding. Mort dives were performed on a regular basisand each netpen received identical standard care practices establishedon this salmon farm. Sampling of fish for growth analyses was performedat either 42 days (APS Process I) or 120 days or greater (Control) fish.In both cases, fish were removed from the netpens and multiple analysesperformed as described below.

All calculations to obtain feed conversion ratio (FCR) or specificgrowth rate (SGR) and growth factor (GF3) were performed using standardaccepted formulae (Willoughby, S. Manual of Salmonid Farming BlackwellScientific, Oxford UK 1999) and established measurements of degree daysfor the Blue Hill Bay site as provided in Table 2 below. A degree day isa measure of the number of days that a month in which a salmon can grow.It is calculated by multiplying the number of days in a month by theamount of degrees in Celsius.

TABLE 2 Degree days for Blue Hill Bay Salmon Aquaculture Site MonthDegree Days Jan 60 Feb 30 Mar 15 April 120 May 210 June 300 July 390 Aug450 Sept 420 Oct 360 Nov 240 Dec 180

Table 3 displays data obtained after seawater transfer of Control S1smolt. Smolt ranging from 75–125 gm were placed into 3 independentnetpens and subjected to normal farm practices demonstratedcharacteristics typical for present day salmon aquaculture in Maine.Significant mortalities (average 3.3%) were experienced after transferinto cool (10° C.) seawater and full feeding was achieved only after asignificant interval (˜56 days) in ocean netpens. As a result, theaverage SGR and GF3 values for these 3 netpens were 1.09 and 1.76respectively for the 105–121 day interval measured.

In contrast to the immediate transfer of Control S1 smolt as describedabove to ocean netpens (Table III), a total of 10,600 S1 smoltpossessing an average size of 63.6 grams were transported on 11 May 2000from the Deblois freshwater hatchery to the research facility. Whilebeing maintained in standard circular tanks, these fish were held for atotal of 45 days at an average water temperature of 11° C. and weresubjected to APS Process I. During this interval, smolt mortality wasonly 64 fish (0.6%). As a matched control for the APS Process I fish, asmaller group of control fish (n=220) were held under identicalconditions but did not receive the APS Process I treatment. Themortalities of these control fish were minimized by the holdingtemperature of 10° C. and were equivalent to treated smolts prior totransfer to seawater.

TABLE 3 Characteristics of St. John S1 smolt subjected to immediateplacement in ocean netpens after transport form the freshwater hatcherywithout APS technology (the Control fish) Netpen Number #17 #18 #10Total Fish 51,363 43,644 55,570 Mean Date of May 1, 2000 May 5, 2000 May14, 2000 Seawater Transfer Average Size at (117.6) Transfer (grams)100–125 75–100 75–100 Mortalities after 30 1,785; 3.5% 728; 1.7% 2503;4.5% days (# and % total) Time to achieve full 68 days 48 days 50 daysfeeding after transfer Interval between 121 120 105 netpen placement andanalysis Average size at Analysis Weight (gram) 376.8 ± 74 305.80 + 64298.90 + 37.40 Length (cm) 33.4 + 1.9 28.30 + 9.0 30.40 + 1.17 ConditionFactor (k) 1.02 1.34 1.06 SGR 0.96 1.10 1.17 during initial 120 days

During the 45 day interval when S1 smolts were receiving APS Process I,fish grew an average of 10 grams and thus possessed an average weight of76.6 gm when transferred to an ocean netpen. The actual smolt transferto seawater occurring on 26 Jun. 2000 was notable for the unusual vigorof the smolt that would have normally been problematic since this timeis well past the normal window for ocean placement of smolt. The oceantemperature at the time of APS Process I smolt netpen placement was15.1° C. In contrast to the counterpart S1 smolts subjected to standardindustry practices described above, APS Process I smolts fed vigorouslywithin 48 hours of ocean placement and continued to increase theirconsumption of food during the immediate post-transfer period. Themortality of APS Process I smolts was low (6.1%) during initial 50 daysafter ocean netpen placement and two thirds of those mortalities weredirectly attributable to scale loss and other physical damage incurredduring the transfer process itself.

In contrast, corresponding control fish (held under identical conditionswithout APS Process I treatment) did not fare well during transfer tothe netpen (17% transfer mortality) and did not feed vigorously at anytime during the first 20 days after ocean netpen placement. This smallernumber of control fish (176) were held in a smaller (1.5 m×1.5 m×1.5 m)netpen floating within the larger netpen containing APS Process Ismolts. Their mortality post-ocean netpen placement was very high at 63%within the 51 day interval.

Both APS Process I and control smolts were fed on a daily basis in amanner identical to that experienced by the Industry Standard Fish shownon Table 3. APS Process I fish were sampled 51 days after their seawaterplacement and compared to the Industry Standard smolts shown on Table 2.As shown in Table 4, comparison of their characteristics revealsdramatic differences between Industry Standard smolts vs APS Process I.

TABLE 4 Comparison of the characteristics of St. John S1 APS Process ISmolts subjected to APS treatment and then placed in ocean netpens vscorresponding industry standard smolts. Averaged Industry APS Process IStandard Data from Table Smolts 3 in this Example Total Fish 10,600150,577 Mean Date of Seawater Jun. 26, 2000 May 7, 2000 Average Size atTransfer 76.6 95.8 (grams) Mortalities after 30 days 648; 6.1% 21,618;14.3% (# and %) Time to achieve full 48 hrs 56 days Feeding aftertransfer Interval between netpen 51 115 placement and analysis Averagesize at Analysis 175.48 + 50 327.2 Weight (gram) 262.22 + 32 30.7 Length(cm) Condition Factor (k) 0.95 + 0.9 1.14 SGR 1.80 1.09

In summary, notable differences between APS Process I, Control smolt andIndustry Standard smolt include:

1. The mortalities observed after ocean netpen placement were low in APSProcess I (6.1%) vs Control (63%) despite the that fact these fish weretransferred to seawater 1.5 months after the smolt window and into avery high (15.1° C.) ocean water temperature. The mortality of APSProcess I was actually lower than that of Industry Standard smolt(14.3%) transferred to cooler (10° C.) seawater during the smolt window.This characteristic of APS Process I provides for a greater flexibilityin freshwater hatchery operations since placement of APS Process Ismolts are not rigidly confined the conventional “smolt window”currently used in industry practice.

2. The APS Process I fish were in peak condition during and immediatelyafter seawater transfer. Unlike industry standard smolt that required 56days to reach full feeding, the APS Process I smolts fed vigorouslywithin 48 hours. Moreover, the growth rates (SGR 1.8) demonstrated byAPS Process I smolts are significantly greater than both published datafor standard smolt during their initial 50 days after seawater placement(published values (Stradmeyer, L. Is feeding nonstarters a waste oftime. Fish Farmer 3:12–13, 1991; Usher, M L, C Talbot and F B Eddy.Effects of transfer to seawater on growth and feeding in Atlantic salmonsmolts (Salmo salar L.) Aquaculture 94:309–326, 1991) for SGR's rangebetween 0.2–0.8). In fact, the growth rates of APS Process I smolts aresignificantly larger than Industry standard smolts placed on the samesite despite the fact that industry standard smolt were both larger atthe time of seawater placement as well as the fact that their growth wasmeasured 120 days after seawater placement. These data provide evidencethat the APS Process I smolts were not subjected to significantosmoregulatory stress which would prevent them from feeding immediately.

3. The rapid growth of APS Process I smolts immediately upon oceannetpen placement provides for compounding increases in the size ofsalmon as seawater growout proceeds. Thus, it is anticipated that ifIndustry Standard Smolts weighing 112.5 gram were subjected to APSProcess I treatment, placed in ocean netpens and examined at 120 daysafter ocean netpen placement their size would be average 782 graminstead of 377 gram as observed. This provides for more than a doublingin size of fish in the early stages of growout. Such fish would reachmarket size more rapidly as compared to industry standard fish.

FIG. 6 provides data on the characteristics of APS Process I smoltsafter seawater transfer.

Application of the APS Process I to Atlantic Salmon Pre-Adult Fish thatare Smaller than the Industry Standard “Critical Size” Smolt.

A total of 1,400 Landcatch/St John strain fingerlings possessing anaverage weight of 20.5 gram were purchased from Atlantic Salmon of MaineInc., Quossic Hatchery, Quossic, Me., USA on 1 Aug. 2000. Thesefingerlings were derived from a egg hatching in January 2000 andconsidered rapidly growing fish. They were transported to the treatmentfacility using standard conventional truck transport. After theirarrival, these fingerlings were first placed in typical freshwatergrowout conditions for 14 days. These fingerlings were then subjected toAPS Process I for a total of 29 days while being exposed to a continuousphotoperiod. The APS Process I were then vaccinated with the LipogenForte product (Aquahealth LTD.) and transported to ocean netpens byconventional truck transport and placed into seawater (15.6° C.) ineither a research ocean netpen possessing both a predator net as well asnet openings small enough (0.25 inch) to prevent loss of these smallerAPS Process I smolts. Alternatively, APS Process I smolts were placed incircular tanks within the laboratory. Forty eight hours after sea watertransfer, APS Process I smolts were begun on standard moist (38%moisture) smolt feed (Connors Bros.) that had been re-pelletized due tothe necessity to provide for smaller size feed for smaller APS Process Ismolts, as compared to normal industry salmon. In a manner identical tothat described for 70 gram smolts above, the mortality, feedconsumption, growth and overall health of these 30 gram APS Process Ismolts were monitored closely.

FIG. 7 displays the characteristics of a representative sample of alarger group of 1,209 APS Process I smolts immediately prior to theirtransfer to seawater. These parameters included an average weight of26.6+8.6 gram, length of 13.1+1.54 cm and condition factor of 1.12+0.06.After seawater transfer, APS Process I smolts exhibited a low initialmortality despite the fact that their average body weight is 26–38% ofindustry standard 70–100 gram S0–S1 smolts. As shown in Table 5, APSProcess I smolts mortality within the initial 72 hr after seawaterplacement was 1/140 or 0.07% for the laboratory tank. Ocean netpenmortalities after placement of APS Process I smolts were 143/1069 or13.4%. FIG. 7 shows representative Landcatch/St John strain APS ProcessI smolts possessing a range of body sizes that were transferred toseawater either in ocean netpens or corresponding laboratory seawatertanks. APS Process I smolts possess a wide range of sizes (46.8–5.6gramsbody weight) with an average body weight of 26.6 gram.

TABLE 5 Characteristics and survival of APS Landcatch/St. JohnSupersmolts I after their placement into seawater in either an APSlaboratory tank or ocean netpen. Laboratory Tank Ocean Netpen Total Fish140 1,069 Date of Seawater Transfer Sep. 5, 2000 (40); Sep. 12, 2000Sep. 12, 2000 (100) Average Size at Transfer 26.6 26.6 (gram) Totalmortalities after 4 1; 0.7% 143; 13.4% days (# and % total) % mortalityof fish 0; 0.0% 4; 0.4% weighing 25 gm and above Time to achieve feeding48 hrs 72 hrs

FIG. 8 shows a comparison of the distributions of body characteristicsfor total group of Landcatch/St John APS Process I smolts vs.mortalities 72 hr after seawater ocean netpen placement. Length and bodyweight data obtained from the 143 mortalities occurring after seawaterplacement of 1,069 APS Process I smolts were plotted on data obtainedfrom a 100 fish sampling as shown previously in FIG. 7. Note that themortalities are distributed among the smaller fish within the larger APSProcess I netpen population.

Length and weight measurements for all mortalities collected from thebottom of the ocean netpen were compared to the distribution of APSProcess I smolt body characteristics obtained from analysis of arepresentative sample prior shown in FIG. 8. The data show that themortalities occurred selectively amongst APS Process I smolts possessingsmall body sizes such that the mean body weight of mortalities was 54%of the mean body weight of the total transfer population (14.7/27 gramor 54%). Thus, the actual mortality rates of APS Process I smoltsweighing 25–30 gram is 0.4% (4/1069) and those weighing 18–30 gram is2.9% (31/1069).

Application of APS Process I to Trout Pre-Adult Fish that are Smallerthan the Industry Standard “Critical Size” Smolt.

Table 6 displays data on the use of the APS Process I on small (3–5 gm)rainbow trout. Juvenile trout are much less tolerant of abrupt transfersfrom freshwater to seawater as compared to juvenile Atlantic salmon. Asa result, many commercial seawater trout producers transfer their fishto brackish water sites located in estuaries or fresh water lenses orconstruct “drinking water” systems to provide fresh water for troutinstead of the full strength seawater present in standard ocean netpens.After a prolonged interval of osmotic adaptation, trout are thentransferred to more standard ocean netpen sites to complete theirgrowout cycle. In general, trout are transferred to these ocean sitesfor growout at body weights of approximately 70–90 or 90–120 gram.

TABLE 6 Comparison of the Survival of Rainbow Trout (3–5 gm) in SeawaterAfter Various Treatments. Percent Survival of Fish¹ Constant 14 Constant23 Hours Post Constant 14 day day Seawater Control day PhotoperiodPhotoperiod + Transfer Freshwater Photoperiod APS Process APS Process 0100 100 100 100 24 0 25 80 99 48 0 70 81 72 40 68 96 30 58 120 30 46Number of 10 20 30 80 Fish Per Experiment ¹Survival percentagesexpressed as rounded whole numbers

A total of 140 trout from a single pool of fish less than 1 yr old weredivided into groups and maintained at a water temperature of 9–13° C.and pH 7.8–8.3 for the duration of the experiment described below. Whencontrol freshwater rainbow trout are transferred directly into seawater,there is 100% mortality within 24 hr (Control Freshwater). Exposure ofthe trout to a constant photoperiod for 14 days results in a slightimprovement in survival after their transfer to seawater. In contrast,exposure of trout to APS Process I for either 14 days or 23 days resultsin significant reductions in mortalties after transfer to seawater suchthat 30% and 46% of the fish respectively have survived after a 5 dayinterval in seawater. These data demonstrate that application of the APSProcess I increases in the survival of pre-adult trout that are lessthan 7% of the size of standard “critical size” trout produced bypresent day industry standard techniques.

Application of the APS Process I to Arctic Char Pre-Adult Fish that areSmaller than the Industry Standard “Critical Size” Smolt.

Although arctic char are salmonids and anadromous fish, their toleranceto seawater transfer is far less as compared to either salmon or trout.FIG. 9 shows the results of exposure of smaller char (3–5 gm) to the APSProcess I for a total of 14 and 30 days. All fish shown in FIG. 9 wereexposed to a continuous photoperiod. Transfer of char to seawaterdirectly from freshwater results in the death of all fish within 24 hr.In contrast, treatment of char with the APS Process I for 14 and 30 daysproduces an increase in survival such that 33% (3/9) or 73% (22/30)respectively are still alive after a 3 day exposure. These datademonstrate that the enhancement of survival of arctic char that areless than 10% of the critical size as defined by industry standardmethods after their exposure to the APS Process I followed by transferto seawater.

FIG. 9 shows a comparison of survival of arctic char after varioustreatments. A single group of arctic char (3–5 gm were obtained fromPierce hatcheries (Buxton, Me.) and either maintained in freshwater ortreated with the APS Process I prior to transfer to seawater.

SECTION II: The Use of the APS Process II to Permit Successful Transferof 10–30 Gram Smolt into Seawater Netpens and Tanks.

The APS Process II protocol is utilized to treat pre-adult anadromousfish for placement into seawater at an average size of 25–30 gm or less.This method differs from the APS Process I protocol by the inclusion ofL-tryptophan in the diet of pre-adult anadromous fish prior to theirtransfer to seawater. APS Process II further improves the osmoregulatorycapabilities of pre-adult anadromous fish and provides for still furtherreductions in the “critical size” for Atlantic salmon smolt transfers.In summary, APS Process II reduces the “critical size” for successfulseawater transfer to less than one fifth the size of the present dayindustry standard SO smolt.

Application of APS Process II to Atlantic Salmon Fingerlings:

St John/St John strain pre-adult fingerlings derived from a January 2000egg hatching and possessing an average weight of 0.8 grams werepurchased from Atlantic Salmon of Maine Inc. Kennebec Hatchery, KennebecMe. on 27 Apr. 2000. These fish were transported to the treatmentfacility using standard conventional truck transport. After theirarrival, these parr were first grown in conventional flow throughfreshwater growout conditions that included a water temperature of 9.6°C. and a standard freshwater parr diet (Moore-Clark Feeds). On 17 Jul.2000, fingerlings were begun on APS Process II for a total of 49 dayswhile being exposed to a continuous photoperiod. APS Process II smoltswere then vaccinated with the Lipogen Forte product (Aquahealth LTD.) onDay #28 (14 Aug. 2000) of APS Process II treatment. APS Process IIsmolts were size graded prior to initiating APS Process II as well asimmediately prior to transfer to seawater. St John/St John APS ProcessII smolts were transported to ocean netpens by conventional trucktransport and placed into seawater (15.2° C.) in either a single oceannetpen identical to that described for placement of APS Process I smoltsor into laboratory tanks (15.6° C.) within the research facility.

FIG. 10 shows representative St. John/St John strain APS Process IIsmolts possessing a range of body sizes were transferred to seawatereither in ocean netpens or corresponding laboratory seawater tanks. Notethat these APS Process II smolts possess a wide range of body weights(3.95–28 gram) that comprised an average body weight of 11.5 gram. FIG.10 shows the characteristics of St. John/St John APS Process II smolts.The average measurements of these St. John/St. John APS Process IIsmolts included a body weight of 11.50+5.6 gram, length of 9.6+1.5 cmand condition factor of 1.19+0.09. The data displayed in Table 7 showsthe outcomes for two groups of APS Process II smolts derived from asingle production pool of fish after their seawater transfer into eitherlaboratory tanks or ocean netpens. Although important variables such asthe water temperatures and transportation of fish to the site ofseawater transfer were identical, these 2 groups of APS Process IIsmolts experienced differential post seawater transfer mortalities after5 days into laboratory tanks (10% mortality) and ocean netpens (37.7%mortality).

TABLE 7 Characteristics and survival of APS St. John/St. JohnSuperSmolts II after their placement into seawater in either alaboratory tank or ocean netpen. Laboratory Tank Ocean Netpen Total Fish100 1,316 Seawater Transfer Date Aug. 31, 2000 Sep, 5, 2000 WaterTemperature (° C.) 15.6 15.6 Size at Transfer (gram) 11.5 11.5 TotalMortalities after 5 10; 10% 496; 37.7% days (# and % total) %mortalities weighing 13 0; 0% 1; 0.08% grams or greater Time to achievefeeding 48 hrs 48 hrs after transfer

No apparent problems were observed with the smaller (10–30gram) APSProcess II smolts negotiating the conditions that exist within theconfines of their ocean netpen. This included the lack of apparentproblems including the ability to school freely as well as the abilityto swim normally against the significant ocean currents that arecontinuously present in the commercial Blue Hill Bay salmon aquaculturesite. While these observations are still ongoing, these data do notsuggest that the placement and subsequent growth of APS Process IIsmolts in ocean netpens will be comprised because of lack of ability ofthese pre-adult anadromous fish to swim against existing ocean currentsand therefore be unable to feed or develop properly.

FIG. 11 compares characteristics of survivors and mortalities of APSProcess II smolts after seawater transfer to either laboratory tanks(FIG. 11A) or ocean netpens (FIG. 11B). FIG. 11A data are derived fromanalyses of 100 APS Process II smolts transferred to seawater tank whereall fish were killed and analyzed on Day 5. In contrast, FIG. 11Bdisplays only mortality data from ocean netpen. In both cases, onlysmaller APS Process II smolts experienced mortality. Note differences inY axis scales of FIGS. 11A–B.

Comparison of the average body size of those APS Process II smolts thatsurvived seawater transfer vs. those APS Process II smolts that diedshows that unsuccessful APS Process II smolts possessed significantlysmaller body weights as compared to average body size of whole APSProcess II smolt transfer group. Thus, the average weight of mortalitiesin laboratory tank (5.10+2.2 gram) and ocean netpen (6.46+1.5 gram) are44% and 56% respectively the value of the average body weight possessedby the entire transfer cohort (11.5 gram). In contrast, the mortalitiesof APS Process II smolts with body weights greater than 13 gram is 0/100in the laboratory tank and 1/1316 or 0.076% for ocean netpens. Together,these data demonstrate that APS Process II is able to redefine the“critical size” of Atlantic salmon smolts from 70–100 gram toapproximately 13 gram.

Application of the APS Process II to Rainbow Trout

Expansion of trout farming has been hampered by several factors. Theseinclude the fact that juvenile trout are much less tolerant of abrupttransfers from freshwater to seawater as compared to juvenile Atlanticsalmon. As a result, many commercial seawater trout producers transfertheir fish to brackish water sites located in estuaries or fresh waterlenses or construct “drinking water” systems to provide fresh water fortrout instead of the full strength seawater present in standard oceannetpens. After a prolonged interval of osmotic adaptation, trout arethen transferred to more standard ocean netpen sites to complete theirgrowout cycle. In general, trout are transferred to these ocean sitesfor growout at body weights of approximately 70–90 or 90–120 gram.

A total of 2,000 Donaldson strain trout with an average weight of 18gram were obtained from a local commercial hatchery source (Pine TreeTrout Farm, Sanford, Me., USA). They were derived from a December 1999egg hatching and were transferred from freshwater to the APS Process IIat 11–12° C. while being exposed to a continuous photoperiod. The totalduration of APS Process II treatment was 35 days (21 Jun.–26 Jul.,2000). After being vaccinated using Lipogen Forte (Aquahealth LTD),trout were transferred directly to a research netpen containing fullstrength seawater at 15.6° C. using standard transfer procedures asdescribed for Atlantic salmon above. The average weight for the totalgroup of Trout APS Process II was 22.7 gram as shown on Table 8.

Mortality counts performed identically to those described for Atlanticsalmon transfers revealed a total of 513/1190 or 43.1% during theinitial 5-day interval. The average body weight of these mortalities was15.5+1.5 gram as shown on FIG. 12. In a manner similar to that displayedby Atlantic salmon APS Process I and II smolts, mortalities occurredamongst the smaller trout APS Process II smolts while the larger fishexhibited little or no deaths. Thus, the average body weight for themortality population was 15.5 gram or 68.3% of the value for totalpopulation of trout transferred to seawater. Feeding of trout wasobserved upon offering moist diet feed at 48 hours after placement infull strength seawater.

TABLE 8 Characteristics and Survival of Donaldson Rainbow TroutSuperSmolts II After Their Direct Placement into Full Strength Seawaterin APS Ocean Netpen. Trout APS SuperSmolts II Total Fish 1,190 Date ofSeawater Transfer Jul. 25, 2000 Average Size at Transfer (grams) 22.7grams Mortalities after 5 days (# and % totla) 513; 43.1% Average Sizeof Morts (grams) 15.5 ± 1.52 Average Size of Survivors (grams) 29.35 +8.3 Time to achieve feeding after transfer 48 hours

FIG. 12 shows a distribution of body weights and lengths amongstmortalities of trout APS Process II smolts during the initial 5 daysafter transfer to ocean netpens. Note that the average weight of these515 mortalities is 15.5+1.5 gram.

In summary, these data demonstrate that the benefits of the presentinvention are not confined to Atlantic salmon but also occur usingrainbow trout. Application of the APS Process II smolts process hassignificantly reduced the “critical size” of rainbow trout for directseawater transfer to approximately 30 gram. Moreover, it has eliminatedthe necessity for the transfer of rainbow trout into brackish water.Thus, application of the APS Process II promises to greatly expand thepossible number of sites that can be utilized for full strength seawatertransfer of rainbow trout.

Quantitation of Feeding and Growth of APS Process I and II Smolts afterSeawater Transfer:

Landcatch/St John APS Process I smolts were offered food beginning 48 hrafter their seawater transfer to either laboratory tanks or oceannetpens. While these APS Process I smolts that were transferred tolaboratory tanks began to feed after 48 hr, those fish transferred toocean netpens were not observed to feed substantially until 7 days. Tovalidate these observations, the inventors performed direct visualinspection of the gut contents from a representative sample of 49 APSProcess I smolts 4 days after their seawater transfer to laboratorytanks. A total of 21/49 or 42.9% possessed food within their gutcontents at that time.

The St John/St John APS Process II smolts fed vigorously when firstoffered food 48 hrs after their seawater transfer regardless of whetherthey were housed in laboratory tanks or ocean netpens. An identicaldirect analysis of APS Process II smolts gut contents performed asdescribed above revealed that 61/83 or 73.5% of fish were feeding 4 daysafter transfer to seawater. The vigorous feeding activity of APS ProcessII smolts in an ocean netpen as well as laboratory tanks occurred. Takentogether, these data suggest that APS Process I and II smolts do notsuffer from a prolonged (20–40 day) interval of poor feeding afterseawater transfer as is notable for the much larger industry standardAtlantic salmon smolts not treated with the process.

Due to the fact that these groups of pre-adult Atlantic salmon subjectedto either APS Process I or APS Process II have been transferred veryrecently to seawater, it is not possible to report on their ocean netpengrowth rates, as shown for larger S1 smolts (Table 4). However, APS hasquantified the growth rates of identical fish treated with either APSProcess I or II within laboratory seawater tanks. As shown in Table 9,both Atlantic salmon treated with APS Process I or II grow rapidlyduring the initial interval after transfer to seawater. In contrast toindustry standard smolt weighing 70–100 grams that eat poorly and thushave little or no growth during their first 20–30 days after transfer toseawater, pre-adult Atlantic salmon receiving APS Process I or II bothexhibited substantial weight gains and growth despite the fact that theyare only 27–38% (APS Process I) and 12–16% (APS Process II) for thecritical size of industry standard smolts.

TABLE 9 Comparison of Growth Rates of Pre-adult Atlantic Salmon Exposedto either APS Process I or APS Process II and Placed in Laboratory TanksDuring Initial Interval After Seawater Transfer APS Process I APSProcess II Number of Fish 140 437 Weight at Placement into  26.6  11.50Seawater Days in Seawater  22  21 Placement Weight  26.6*  13.15*Corrected for Mortalities Weight after Interval in  30.3  15.2 SeawaterWeight Gained in  3.75  2.05 Seawater SGR (% body weight/day)  0.60 0.68 FCR  1.27  2.04 *Weight gain corrected for selective mortalitiesamongst smaller fish (4/140 or 2.9% APS Process I; 103/437 or 23.6% APSProcess II)

Example 3 Exposure of Salmon Smolts to Ca2+ and Mg2+ IncreasesExpression of PVCR

In smolts that were exposed to 10 mM Ca²⁺ and 5.2 mM Mg²⁺, theexpression of PVCR was found to increase in a manner similar to that insmolts that are untreated, but are transferred directly to seawater.

Tissues were taken from either Atlantic salmon or rainbow trout, afteranesthesitizing the animal with MS-222. Samples of tissues were thenobtained by dissection, fixed by immersion in 3% paraformaldehyde,washing in Ringers then frozen in an embedding compound, e.g., O.C.T.™(Miles, Inc., Elkahart, Ind., USA) using methylbutane cooled on dry ice.After cutting 8 micron thick tissue sections with a cryostat, individualsections were subjected to various staining protocols. Briefly, sectionsmounted on glass slides were: 1) blocked with goat serum or obtainedfrom the same species of fish, 2) incubated with rabbit anti-CaRantiserum, and 3) washed and incubated with peroxidase-conjugatedaffinity-purified goat antirabbit antiserum. The locations of the boundperoxidase-conjugated goat antirabbit antiserum were visualized bydevelopment of a rose-colored aminoethylcarbazole reaction product.Individual sections were mounted, viewed and photographed by standardlight microscopy techniques. The methods used to produce anti-PVCRantiserum are described below.

The results are shown in FIGS. 13A–13G, which are a set of sevenphotomicrographs showing immunocytochemistry of epithelia of theproximal intestine of Atlantic salmon smolts using anti-PVCR antiserum,and in FIG. 14, which is a Western blot of intestine of a salmon smoltexposed to Ca2+- and Mg2+-treated freshwater, then transferred toseawater. The antiserum was prepared by immunization of rabbits with a16-mer peptide containing the protein sequence encoded by the carboxylterminal domain of the dogfish shark PVCR (“SKCaR”) (Nearing, J. et al.,1997, J. Am. Soc. Nephrol. 8:40A). Specific binding of the anti-PVCRantibody is indicated by aminoethylcarbazole (AEC) reaction product.

FIGS. 13A and 13B show stained intestinal epithelia from smolts thatwere maintained in freshwater then transferred to seawater and held foran interval of 3 days. Abundant PVCR immunostaining is apparent in cellsthat line the luminal surface of the intestine. The higher magnification(1440×) shown in FIG. 13B displays PVCR protein localized to the apical(luminal-facing) membrane of intestinal epithelial cells. The pattern ofPVCR staining is localized to the apical membrane of epithelial cells(small arrowheads) as well as membranes in globular round cells(arrows). FIG. 13C shows stained intestinal epithelia from arepresentative smolt that was exposed APS Process I and maintained infreshwater containing 10 mM Ca2+ and 5.2 mM Mg2+ for 50 days. Note thatthe pattern of PVCR staining resembles the pattern exhibited byepithelial cells displayed in FIGS. 13A and 13B including apicalmembrane staining (small arrowheads) as well as larger globular roundcells (arrows). FIG. 13D shows a 1900× magnification of PVCR-stainedintestinal epithelia from another representative fish that was exposedto the APS Process I and maintained in freshwater containing 10 mM Ca2+and 5.2 mM Mg2+ for 50 days and fed 1% NaCl in the diet. Again, smallarrowhead and arrows denote PVCR staining of the apical membrane andglobular cells respectively. In contrast to the prominent PVCR stainingshown in FIGS. 13A–D, FIGS. 13E (1440×) and 6F (1900×) show staining ofintestinal epithelia from two representative smolt that were maintainedin freshwater alone without supplementation of Ca2+ and Mg2+ or dietaryNaCl. Both 13E and 13F display a marked lack of significant PVCRstaining. FIG. 13G (1440×) shows the lack of any apparent PVCR stainingupon the substitution of preimmune anti-PVCR antiserum on a sectioncorresponding to that shown in FIG. 13A where anti-PVCR antiserumidentified the PVCR protein. The lack of any PVCR staining is a controlto demonstrate the specificity of the anti-PVCR antiserum under theseimmunocytochemistry conditions.

The relative amount of PVCR protein present in intestinal epithelialcells of freshwater smolts (FIGS. 13E and 13F) was negligible as shownby the faint staining of selected intestinal epithelial cells. Incontrast, the PVCR protein content of the corresponding intestinalepithelial cells was significantly increased upon the transfer of thesesmolts to seawater (FIGS. 13A and 13B). Importantly, the PVCR proteincontent was also significantly increased in the intestinal epithelialcells of smolts maintained in freshwater supplemented with Ca2+ and Mg2+(FIGS. 13C and 13D). The AEC staining was specific for the presence ofthe anti-PVCR antiserum, since substitution of the immune antiserum bythe preimmune eliminated all reaction product from intestinal epithelialcell sections (FIG. 13G).

To further demonstrate the specificity of the anti-CaR antiserum torecognize salmon smolt PVCRs, FIG. 14 shows a Western blot of intestinalprotein from salmon smolt maintained in 10 mM Ca2+, 5 mM Mg2+ and fed 1%NaCl in the diet. Portions of the proximal and distal intestine werehomogenized and dissolved in SDS-containing buffer, subjected toSDS-PAGE using standard techniques, transferred to nitrocellulose, andequal amounts of homogenate proteins as determined by both protein assay(Piece Chem. Co, Rocford, Ill.) as well as Coomassie Blue staining wereprobed for presence of PVCR using standard western blotting techniques.The results are shown in the left lane, labeled “CaR”, and shows a broadband of about 140–160 kDa and several higher molecular weight complexes.The pattern of PVCR bands is similar to that previously reported forshark kidney (Nearing, J. et al., 1997, J. Am. Soc. Nephrol. 8:40A) andrat kidney inner medullary collecting duct (Sands, J. M. et al., 1997,J. Clin. Invest. 99:1399–1405). The lane on the right was treated withthe preimmune anti-PVCR antiserum used in FIG. 13G, and shows a completelack of bands. Taken together with immunocytochemistry data shown inFIG. 13, this immunoblot demonstrates that the antiserum used isspecific for detecting the PVCR protein in salmon.

Example 4 Exposure of Trout Fingerlings to Ca2+ and Mg2+ IncreasesExpression of PVCRs

Development of specific ion transport capabilities in epithelial cellsof gill, kidney and intestinal tissues are important to survival ofpre-adult anadromous fish if they are to survive transfer to seawater.To determine if alterations in the PVCRs accompanied the increase introut fingerling survival in seawater, immunoblotting andimmunocytochemistry was performed on samples from the fingerlings as wasdone for the salmon smolt tissues. The results are shown in FIGS. 15, 16and 19.

FIG. 15 is an immunoblot of intestinal tissue from trout fingerlings.Anti-CaR antiserum identifies multiple bands that are specific for PVCRstaining as determined by comparison of immune (lane marked CaR) vs.preimmune (lane marked pre-immune). Prominent among these bands includesa broad band of 120–160 kDa, together with larger molecular weightcomplexes present above these bands from both intestine and gill tissue.

FIGS. 16A, 16B, 16C, 16D, 16E, 16F, 16G and 16H are a set of eightphotomicrographs showing immunocytochemistry of epithelia of theproximal intestine of rainbow trout using anti-PVCR antiserum. FIGS.16A, 16C and 16E show samples from trout maintained in freshwater alone,while FIGS. 16B, 16D, 16F, 16G and 16H show samples from troutmaintained in freshwater supplemented with 10 mM Ca2+ and 5.2 mM Mg2+and fed a 1% NaCl diet. Proximal intestinal segments are shown in FIGS.16A–16D, and 16G–16H, while distal intestinal segments are shown inFIGS. 16E–16F. FIGS. 16A–16F were treated with immune rabbit anti-CaRantiserum, washed, and developed with horseradish peroxidase-conjugatedgoat anti-rabbit antiserum using an aminoethylcarbazole (AEC) reaction.While FIGS. 16A, 16C and 16E display little or no PVCR staining, FIGS.16B, 16D and 16F show significant PVCR staining that is present on theapical membrane of cells lining the intestinal lumen (small arrowheads)as well as larger globular round cells (arrows). In contrast to sectionsexposed to immune anti-PVCR antiserum, FIG. 16H was treated withpre-immune rabbit anti-CaR antiserum and thus do not contain the coloredAEC reaction product. These data indicate this method specificallydetects PVCR protein bound to the anti-PVCR antiserum. FIG. 16G wasstained directly with Alcian blue (Sheehan, D. C. et al., 1980, Theoryand Practice of Histochemistry, Battelle Press, Columbus, Ohio, USA) tolocalize mucin-producing epithelial cells that are present in intestine.Note the appearance of cells staining for PVCR protein in FIGS. 13D(denoted by small arrows) display a similar morphological appearance tothose stained with Alcian blue in FIG. 13G. These data suggest that PVCRare expressed by mucin producing cells in the intestine where PVCRsignaling actions modulate mucin production in the intestine.

Immunocytochemistry of intestinal tissue shows that the content of PVCRprotein is different in trout maintained in freshwater alone (FIGS. 16A,16C and 16E) vs. freshwater supplemented with Ca2+ and Mg2+ and troutfed a NaCl supplemented diet (FIGS. 16B, 16D, 16F). Normally infreshwater, CaR expression is low in either proximal (FIGS. 15A and 16C)or distal (FIG. 16E) sections of intestine. However, PVCR expression issignificantly increased in both proximal (FIGS. 16B and 15D) and distalsegments (FIG. 15F) after exposure to freshwater supplemented with 10 mMCa2+ and 5.2 mM Mg2+ and feeding of NaCl supplemented diet.

While PVCR protein is localized to several regions of multiple cells,the presence of intense staining on the apical membranes of intestinalepithelial cells (small arrowheads) as well as occasional rounded cells(large arrowheads) are identical to data localizing PVCR protein in boththe dogfish shark (Nearing, J. et al., 1997, J. Am. Soc. Nephrol.8:40A), as well as rat kidney inner medullary collecting duct (IMCD)(Sands, J. M. et al., 1997, J. Clin. Invest. 99:1399–1405). As describedabove for Atlantic salmon smolts, the apical PVCR in trout intestine isinduced by increases in luminal Ca2+ and Mg2+ concentrations, andthereby regulates the NaCl-mediated recovery of water from intestinalcontents. This recovery is important to the survival of marine fish(Evans, D. H., 1993, “Osmotic and Ionic Regulation,” in: The Physiologyof Fishes, ed. D. H. Evans, CRC Press, Boca Raton, Fla., USA, Chapter11, pp. 315–341), as it replaces osmotic water losses that occur via theskin and gill.

The anti-PVCR staining of rounded cells, which are interspersedthroughout the larger intestinal epithelial cells (FIG. 16D) is alsoconsistent with these cells corresponding to mucin-producing cells whichare known to stain intensely with Alcian Blue (Sheehan, D. C. et al.,1980, Theory and Practice of Histochemistry, Battelle Press, Columbus,Ohio, USA) (FIG. 16G).

FIG. 17 shows a representative immunoblot that compares the overalllevels of PVCR content of protein homogenates prepared from gill tissueof trout using the same anti-PVCR antiserum as described in FIGS. 13–15.Prior to dissection and homogenation of gill tissue, trout were exposedto 1 of 3 different treatments including either freshwater, freshwaterwith 10 mM calcium and 5.2 mM magnesium with dietary NaClsupplementation or freshwater with dietary NaCl supplementation only. Inthe gill, the anti-PVCR antiserum also identifies a broad 120–140 kDaand a band of large molecular mass (greater than 200 kDa) that aresimilar to those shown in FIGS. 14 and 15. These data are consistentwith molecular masses of CaRs of known structure and similar to thoseobserved in immunoblotting analyses of multiple organisms, including rat(Sands, J. M. et al., 1997, J. Clin. Invest. 99:1399–1405), flounder,and shark (Nearing, J. et al., 1997, J. Am. Soc. Nephrol. 8:40A). Amoderate level of PVCR expression in gill as defined by PVCR reactivebands occurs when trout are maintained in freshwater (freshwater). Theabundance of PVCR protein is increased when trout are exposed to the APSProcess I (Ca, Mg+NaCl suppl. Feed) as shown in the middle lane (Ca,Mg). In contrast, when trout are maintained in freshwater and fed a NaCldiet without exposure to calcium and magnesium in the freshwater, thereis no change in the overall PVCR staining intensity but rather a shiftof PVCR reactivity from the 120 kDa to the larger 200 kDa highermolecular weight band (lane marked salt). These data demonstrate thatexposure of trout to the APS Process I (freshwater containing 10 mMcalcium, 5 mM magnesium and dietary NaCl supplementation) increases PVCRexpression in gill tissue as compared to freshwater alone. Feeding ofNaCl supplement diet while the trout are maintained in freshwater doesnot produce similar increased expression of the PVCR protein.

Example 5 Immunolocalization of Polyvalent Cation Receptor (PVCR) inMucous Cells of Epidermis and in the Brain of Salmon

The skin surface of salmonids is extremely important as a barrier toprevent water gain or loss depending whether the fish is located infresh or seawater. Thus, the presence of PVCR proteins in selected cellsof the fish's epidermal layer would be able to “sense” the salinity ofthe surrounding water as it flowed past and provide for the opportunityfor continuous remodeling of the salmonid's skin based on thecomposition of the water where it is located.

Methods: Samples of the skin from juvenile Atlantic Salmon resident inseawater for over 12 days were fixed in 3% paraformaldehyde dissolved inbuffer (0.1M NaP04, 0.15M NaCl, 0.3M sucrose pH 7.4), manually descaled,rinsed in buffer and frozen at −80° C. for cryosectioning. Ten micronsections were either utilized for immunolocalization of PVCR usinganti-shark PVCR antiserum or stained directly with 1% Alcian Blue dye tolocalize cells containing acidic glycoprotein components of mucous.

Results and Discussion: FIG. 18A shows that salmon epidermis containsmultiple Alcian Blue staining cells present in the various skin layers.Note that only a portion of some larger cells (that containing acidicmucins) stains with Alcian Blue (denoted by the open arrowheads). Forpurposes of orientation, note that scales have been removed so asterisksdenote surface that was previously bathed in seawater. FIG. 18B showsimmunolocalization of salmon skin PVCR protein that is localized tomultiple cells (indicated by arrowheads) within the epidermal layers ofthe skin. Note that anti-PVCR staining shows the whole cell body, whichis larger than its corresponding apical portion that stains with AlcianBlue as shown in FIG. 18A. The presence of bound anti-CaR antibody isindicated by the rose color reaction product. Although formalquantitation has not yet been performed on these sections, it appearsthat the number of PVCR cells is less than the total number of AlcianBlue positive cells. These data indicate that only a subset of AlcianBlue positive cells contain abundant PVCR protein. FIG. 18C of FIG. 18shows the Control Preimmune section where the primary anti-CaR antiserumwas omitted from the staining reaction. Note the absence of rose coloredreaction product in the absence of primary antibody.

These data demonstrate the presence of PVCR protein in discreteepithelial cells (probably mucocytes) localized in the epidermis ofjuvenile Atlantic salmon. From this location, the PVCR protein could“sense” the salinity of the surrounding water and modulate mucousproduction via changes in the secretion of mucous or proliferation ofmucous cells within the skin itself. The PVCR agonists (Ca2+, Mg2+)present in the surrounding water activate these epidermal PVCR proteinsduring the interval when smolts are being exposed to the process of thepresent invention. This “preconditioning” of Atlantic salmon smolts bythe process of the present invention is important to increased survivalof smolts after their transfer to seawater.

Localization of PVCR Protein in Brain of Atlantic Salmon:

The PVCR protein can been specifically localized to the brain stem areaof Atlantic salmon using immunocytochemistry and antibody raised againsta peptide sequence found in the carboxyl terminal of the shark PVCR.These data are consistent with a role for a PVCR in the modulation ofendocrine function as well as appetite control in Atlantic salmon.

Localization of the expression of calcium receptors to specific regionsof the mammalian brain has been determined. While the exact functions ofmammalian CaRs in many regions of the mammalian brain are still unknown,several lines of evidence indicate that CaRs can integrate alterationsin systemic calcium, sodium and water metabolism with modulations inbrain function that include differences in the secretion of hormonessuch as adrenocorticotrophin (ACTH) from the hypothalamus as well asbehavioral changes such as regulation of thirst or eating. Of importanceto disclosure findings detailed below, PVCRs (CaRs) roles in alterationof endocrine function, drinking and appetite in anadromous fishundergoing transfer from freshwater to seawater are important.

In mammalian brain, there is prominent CaR expression in the subfomicalorgan or SFO. The SFO is a key hypothalamic thirst center and isbelieved to play a role in modulation of drinking activity to integratebody calcium and water homeostasis. Stimulation of drinking behavior bysystemic hypercalcemia via stimulation of CaRs located in the SFO isthought to minimize the dehydration produced by alterations in kidneyfunction that blunt the tubular reabsorption of filtered water by thekidney. The equivalent SFO area of the fish brain is not presently beenidentified.

In the mammalian brain, there is CaR expression in the pons area of thebrainstem particularly around the area postrema near the thirdventricle. The area postrema is known to be collection of neuronsbelieved to mediate appetite and has been termed the “nausea center”.From this portion of the mammalian brain, neuronal pathways provide forintegration of sensory input from vestibular function (sensing ofbalance) as well as visual input via pathways from optic nerves andtheir respective nuclei in the brain. This region of the brain isbelieved to be intimately involved in the nausea produced byhypercalcemia as well as the administration of opiates to humans. Theequivalent area postrema of the fish brain is not presently identified.

A combination of physiological and anatomic data provide evidence forthe role of CaRs to integrate a variety of endocrine functions withchanges in the serum calcium levels in humans. Intravenous infusion ofcalcium sufficient to raise serum calcium concentrations causesselective increases in gonadotropic releasing hormones and thyroidreleasing hormone (TRH) as well ACTH that are produced by the anteriorpituitary. The anterior pituitary gland is known to be intimatelyconnected with specific areas of the hypothalamus that express CaRs.

Increases in the serum calcium concentrations of humans cause multiplealterations in both behavior as well as endocrine function. Thus,hypercalcemia causes increased drinking, decreased food consumption andalterations in the circulating levels of specific hypothalamic hormones.As mentioned below, analogous changes in behavior and circulatinghormone levels occur in preadult anadromous fish during smoltficationand transfer from freshwater to seawater.

Methods:

Whole brains obtained from preadult Atlantic salmon (St. John/St JohnAPS Process II smolts) that were subjected to the APS Process II andtransferred to seawater were dissected free of their surroundings andfixed in 3% paraformaldehyde (PFA) in buffer [identical to otherimmunocytochemistry descriptions]. Eight micron sections were cut,attached to glass slides and processed for immunocytochemistry usingeither nonimmune control antiserum or anti-PVCR of dogfish shark.Specific antibody binding was detected by the rose-colored reactionproduct formed from the action of horseradish peroxidase conjugated goatanti-rabbit secondary antiserum and amino ethylcarbazole. Sections wereviewed and photographed using standard light microscopy techniques.

Results and Discussion:

After examining serial sections from multiple preadult Atlantic salmon(average wt. 10.3 gm), there is consistent localization of PVCR proteinin cells localized in 3 distinct regions of the salmon brain. The firstregion of PVCR localization is distinct staining of neurons in the vagallobe region. The second region of PVCR staining is within neurons in thecommissural nucleus of Cajal. Both of these regions of salmon brain areknown to represent important nuclei in the gustatory (sensing food andeating) as well as general visceral activities including esophageal andintestinal motility (processing of food and intestinal contents fornutrient and water reabsorption). Expression of PVCR protein linksalterations in both serum and CNS calcium concentrations to changes ineating and processing of intestinal contents important for anadromousfish adaptation to seawater.

A third site of PVCR localization in salmon brain is the saccusvasculosus where PVCR protein is distributed throughout multiple celltypes. The saccus vasculosus is ovid and localized on the ventralsurface of the brain between the inferior lobes. This structure ishighly vascula ized and contains connections between the cerebral spinalfluid and the vascular space. Moreover, neurons present in the saccusvasculosus possess massive nerve projections that tract to thesubependymal region of the thalamus. The saccus vasculosus systemmodulates the function of centers of the posterior tubercle andperiventricular thalamus. These areas of the brain are immediatelyadjacent to the pituitary gland.

The localization of PVCR protein(s) in the brain of preadult Atlanticsalmon provides evidence that PVCR can be involved in a variety offunctions in the central nervous system of anadromous fish in a mannersimilar to that described above for the mammalian brain. In particular,localization of PVCR to nuclei that are part of the gustatory system inAtlantic salmon indicates that PVCR protein is expressed in neurons thatmodulate appetite similar to that described for the area postrema inmammals. Stimulation of PVCR or alterations in its expression viachanges in the serum calcium, magnesium or sodium concentrations asdemonstrated for Atlantic salmon in this application would then be ableto modulate appetite and food consumption. Alternatively, alterations inthe cerebral spinal fluid concentration of these ions via exchangebetween the CSF and the vascular system can also be involved. SinceAtlantic salmon smolt produced by present day industry standard methodsexperience an interval of profound anorexia after their transfer toseawater, this well known suppression of appetite can be mediatedthrough PVCR signaling mechanisms.

In a similar manner, PVCR signaling pathways can also modulate bothdrinking behavior and pituitary hormone secretion. PVCR proteinexpressed in the saccus vasculosus can provide for both the initiationof the drinking of seawater by Atlantic salmon and can be directlyanalogous to increased drinking in mammals caused by hypercalcemia.Increases in serum calcium, magnesium and sodium concentrations producedby transfer of preadult anadromous fish from freshwater to seawater canalso be the stimulus for increased secretion of hypothalamic hormonessuch as ACTH. ACTH stimulates the secretion of cortisol by the adrenalgland in fish. Cortisol is one hormone that has been shown to be amodulator of ion transport activity and involved in modulation of theparr-smolt transformation in anadromous fish. Modulation of pituitaryactivity via connections between the saccus vasculosus, hypothalamus andthe pituitary can modulate these endocrine changes.

Example 7 Serum Level in Fish Exposed to APS Process I or APS Process II

The data described herein demonstrates that Alterations in theConcentrations of Calcium, Magnesium and NaCl in the Body Fluids ofAnadromous Fish Occur After Seawater Transfer and Excessively HighConcentrations cause or contribute to Post Seawater Transfer Deaths inAnadromous Fish. APS Process II Mimics Seawater Transfer WithoutSubjecting Small Preadult Anadromous Fish to Osmotic Stress. This“Preconditions” Fish thus Allowing Them to be Transferred to Seawater atSignificantly Smaller Sizes and Under Conditions That are Nonpermissiveusing Industry Standard Practices.

PVCRs are present in multiple tissue locations where PVCRs are exposedto surrounding seawater (gills, skin), luminal contents of tubules(kidney, intestine) as well as internal body fluids (brain, endocrinetissue, muscle). When anadromous fish are transferred from fresh toseawater there is an abrupt rise in the external water concentrations ofcalcium, magnesium and NaCl. If the fish absorbs increased amounts ofcalcium, magnesium and NaCl via drinking or osmosis then PVCRs locatedon the apical surfaces of intestinal and kidney epithelial cells will beexposed to increased amounts of these divalent and monovalent ions.These increases in divalent cation concentrations occur since the kidneyis the primary excretory organ for divalent cations and the intestine isthe major water recovery organ for anadromous fish via the processing ofingested seawater. Important for this data disclosure is the fact thatif the concentrations of calcium, magnesium and NaCl increase in theblood and extracellular fluid of fish, then the PVCRs that are bathed inthese body fluids will become stimulated. Alterations in serum calciumand magnesium constitute an actual signaling pathway. In this regard, itis also noteworthy that there are a wide range of “normal” values forserum concentrations of calcium, sodium, magnesium and chloride inanadromous fish. While it has been recognized that steady state serumconcentrations of these ions change with differing salinities, there hasbeen no recognition that these might represent fish with differing PVCR“set points” as described herein.

Current production methods for salmonids depend on the attainment of a“critical size” for preadult fish called smolt to enable them to survivethe transfer from freshwater to seawater.

The production of salmonids for aquaculture is dependent on the abilityfor preadult fish to survive direct transfer from freshwater toseawater. For this process to occur, present day industry methods haveidentified a “critical size” for each species of salmonid. Below thiscritical size, many fish are not able to survive the dramaticalterations in water osmolality and ionic composition. Factors thatcontribute to the ability of “critical size” smolt include specificsurface area to volume ratios as well as the maturity of ionic transportand hormonal mechanisms to cope with the new seawater ionic environment.These mechanisms involve coordinated responses from several organsincluding the gill, gastrointestinal tract, kidney, and skin as well asspecific behavioral changes such as the initiation of drinking behaviorafter seawater exposure. The transfer of a fish from a freshwater toseawater environment constitutes a major challenge to theseosmoregulatory systems that are rapidly remodeled to permit itssurvival. The basic osmoregulatory mechanisms and responses are outlinedbriefly on FIG. 19.

When a fish resides in freshwater, it is surrounded by an aqueousenvironment that possesses a significantly lower ionic and osmoticcontent (Table 11). Due to the osmotic gradient that exists between thebody fluid of the fish and the surrounding environment, the fish isconstantly gaining water that continuously threatens to dilute the moreconcentrated ionic content of the fish's body fluids. As a result, thefreshwater fish do not drink and excrete a copious dilute urine. Toprevent the loss of important body salts into the environment, thegills, gastrointestinal tract as well as kidney tubules engage in activeuptake of ions from either their luminal contents or the surroundingfreshwater.

TABLE 11 Comparison of the Ionic¹ and Osmotic² Composition of Seawaterand Freshwater vs Serum (Blood) of Atlantic Salmon³ Seawater FreshwaterAtlantic Salmon Sodium 450 0.3–5   135–185 Calcium 10 0.07–2   2.5–3.9Magnesium 50 0.04–3   1.0–2.8 Chloride 513 0.23–10   120–138 Sulfate 260.05 <0.02 Osmolality 1050  1–20 330–390 ¹All values expressed asmMoles/Liter. ²Values expressed as mOsmoles/kg H₂O ³Values vary whetherfish is in freshwater or seawater. Range of average values provided.

In contrast, when a fish resides in seawater the surrounding aqueousenvironment possess a significantly larger ionic and osmotic content ascompared to the fish's own body composition (Table 11). As shown in FIG.19, marine salmonids are constantly losing body water content to thesurrounding seawater. In this regard, both the integrity andpermeability of the fish's skin layer are important in reducing thesecutaneous losses to as low as possible. To replace these ongoing waterlosses, the fish drinks seawater and processes it in such a way toretain water and only a portion of its constituent ions. Ingestedseawater is processed by epithelial cells lining the gastrointestinaltract. In this process, the intestinal uptake of water and some NaCl bythe fish is permitted while Ca2+ and Mg2+ are either not absorbed orexcreted by kidney tubules. Absorbed NaCl is pumped from the fish's bodyvia gill epithelial cells.

FIG. 19 compares adaptive changes present in fish in freshwater vsseawater. Specific physiological adaptations present in freshwater fishare shown schematically on the left panel. In contrast, alterations inthese same physiological responses when fish are in seawater are shownon the right.

It is important for the pre-adult anadromous fish to accomplish all ofthese adaptative changes rapidly after transfer from freshwater toseawater. Deployment and maturation of these mechanisms requires thesynthesis of new proteins and remodeling of epithelial cells involved intransepithelial transport. These changes occur in a time scale that willpermit the smolt to survive in its new seawater environment. The smallerthe fish, the larger its surface area/volume ratio. Thus, smaller fishlose their body water more rapidly and have less body water stores tobuffer changes in body ionic composition. As a result, small fishrapidly lose water and they cannot replace this water via drinkingseawater since their ionic removal mechanisms are not mature. As aresult, smaller or nonmature smolts rapidly die of electrolyte and waterimbalances produced by their inability to adapt to the new osmotic andionic environment of seawater. In contrast, larger smolts that arelarger than the “critical size” possess a lower surface area to volumeratio, lose water less rapidly and have more body water to buffer ionicchanges. This larger body size provides them the interval of timenecessary to deploy their more mature ionic transport mechanismsenabling them to survive.

In smolts that are either less than the critical size or possessimmature physiological ion transport mechanisms, the combination of theosmotic removal of water from their bodies coupled with ingestion of ionrich seawater produces specific alterations in body fluid andelectrolyte composition. These changes include: a decrease in total bodywater content, increases in the concentrations of calcium, magnesium andsodium chloride. Abnormally high concentrations of these monovalent anddivalent cations causes a wide range of specific changes in organ andcellular functions including alterations in cellular metabolism andnerve conduction, depression of normal nervous system and muscleactivity as well as cessation of normal ingestion of food and itsdigestion. The abnormal behavior and appearance of highly stresspre-moribund fish after seawater transfer are actually attributable tothe physiological effects of elevated ions including calcium andmagnesium within the body fluids of the fish.

As described herein, measurements of serum calcium, magnesium and sodiumconfirm these data as well as demonstrate that the present inventioncauses a preconditioning of physiological and ionic transport mechanismspermitting the successful seawater transfer of preadult anadromous fishthat are significantly smaller than the critical size as defined bypresent day industry standard methods.

Methods:

Blood was obtained from fish (salmon and trout) via venipuncture intothe caudal sinus and prevented from coagulation by the addition oflithium heparin. The blood was centrifuged at 4,000 rpm for 10 minutesand the resulting serum collected and stored until assay. Calcium andmagnesium concentrations of 2 microliter aliquots of serum werequantified using calcium and magnesium assay kits (Kit #595, #587 SigmaAldrich, St Louis, Mo.) and Na was determined by commercial testing(NorDx Laboratories, Scarborough, Me.) using a Hitachi 747 analyzer.

Results and Discussion:

The APS Process II Mimics Exposure to Seawater Without the Presence of aLarge Osmotic Gradient Between the Fish's Body Fluids and SurroundingHypertonic Seawater.

FIG. 20 shows the changes in serum calcium concentrations in juveniletrout (average body weight approximately 30 gm) subjected to seawatertransfer either directly from treshwater or after exposure to variouscomponents of APS Process II. The average steady state serum calciumconcentration in these trout maintained in freshwater is 2.72+0.16 mM.In contrast, transfer of trout to seawater results in a significant risein serum calcium to approximately 3.80 mM within the initial 24 hr afterseawater transfer. This increase in serum calcium is sustained for aninterval of approximately 108 hr (4.5 days) but then declines to aslightly lower average concentration of 3.20+0.42 mM by 126 hr. Thus,internal PVCRs are exposed to a rise in serum calcium upon transfer offreshwater trout to seawater. The aquatic PVCRs would actually sense andrespond to alterations in calcium this concentration range. Thus, theincrease in serum calcium (a PVCR agonist) likely constitutes a signalfor the initiation of multiple PVCR-activated processes in variousorgans to permitting the survival of juvenile trout in seawater.

Placement of trout in the water mixture of the present invention whichcontains 3 mM calcium and 1 mM magnesium, and feeding the trout astandard freshwater diet (Moore Clarke Feeds) results in no significantincreases in serum calcium as compared to serum calcium values for troutmaintained in freshwater despite the presence of a net inward gradientof calcium from external water mixture (3 mM) to internal body fluids(2.72 mM). Moreover, serum calcium concentrations of trout maintained inthe water mixture are not changed by alterations of the ambientphotoperiod from a normal (10 hr daylight; 14 hr darkness) to continuousdaylight exposure.

FIG. 21 shows increases in serum calcium concentrations induced byfeeding trout maintained in water mixture (3 mM calcium, 1 mM magnesium)a standard freshwater pelleted diet containing additional 1% sodiumchloride (w/w). Feeding of NaCl supplemented diet began immediatelyafter determination of baseline serum calcium concentrations at timezero. Note that serum calcium concentrations became elevated after aninterval of 24 hr. Data points shown represent a total of 5 or moreindependent determinations from a single representative experiment.Values at 24 hr and 72 hr are significantly (p<0.05) increased ascompared to the value at zero time.

In contrast, the feeding of trout maintained in the water mixture of thepresent invention with the identical standard feed except with theaddition of 1% NaCl (weight/weight) produces a significant increase inserum calcium concentrations within 24 hr (FIG. 21). This increase inthe serum calcium concentrations of trout mimics the rise produced bytransfer of trout into seawater(compare FIG. 20 vs. 21). This effect ofdietary NaCl to increase serum calcium levels likely occurs because thefish is obligated to excrete this excess NaCl that it has ingested.Ingestion of this excess NaCl activates the fish's drinking behaviorthereby causing it to ingest water mixture containing 3 mM calcium andthereby increases its body fluid calcium content via the intestinalabsorption of calcium. Ingestion of 1% NaCl alone does not alter serumcalcium concentrations. Thus, the serum calcium concentration of troutmaintained in freshwater (2.72+0.43 n=6) was not altered significantlyafter consumption of feed containing 1% NaCl (w/w) for as long as 30days (2.37+0.25 n=5). These data provide a demonstration that thisprotocol is necessary to achieve increase the serum calciumconcentrations in anadromous fish.

Transfer of Larger Atlantic Salmon Smolts Raised in Freshwater thatPossess the Industry Standard “Critical Size” to Seawater Raises theirSerum Calcium and Sodium Concentrations:

The data displayed in FIG. 20 shows that the mean serum calciumconcentration increases by approximately 40% when trout are transferredfrom freshwater to seawater. The magnitude of this increase isassociated with significant trout mortality (approximately 30–40%) dueto osmoregulatory failure in these fish that are smaller than the“critical size” for trout. In contrast, the magnitude of increases inserum calcium concentrations is smaller (approximately 30% increase)when larger Atlantic salmon smolts that possess the critical size 60–70gms are transferred to seawater (FIG. 22A–B). During this same intervalafter seawater transfer, serum sodium concentrations in these same fishincrease by approximately 17%. Data derived from both trout (FIG. 21)and salmon (FIG. 22A–B) were only collected from fish that exhibited novisible signs of stress (i.e. stressed fish exhibit body discoloration,bizarre swimming behavior or markedly decreased activity levels) duringthis experiment.

FIG. 22 shows alterations in serum calcium (FIG. 22A) and sodium (FIG.22B) after seawater transfer of S1 Atlantic salmon smolt that possessthe critical size as defined by standard present day practices. Eachdata point represents the mean±S.D of 5–10 independent determinations

FIGS. 23A–B show post seawater transfer values for serum calcium,magnesium and sodium obtained from a cohort of 80 S1 Atlantic salmonsmolt identical to that shown in FIG. 22 after a total of 45 days oftreatment with APS Process I (3 mM Ca²⁺, 1 MM Mg²⁺ in water) and 7% NaCldietary supplement in food. Note that the initial serum calcium in fishexposed to APS Process I is slightly larger (2.5 vs 3.0 mM) and changesin serum concentrations of calcium and sodium are similar to thosedisplayed in FIG. 22. Moreover, calcium and magnesium do not undergodramatic increases during the initial 120 hr interval as these fish aretransferred from calcium/magnesium water mixture to seawater. Incontrast, the serum sodium concentration increases approximatley 12%(178.8 mM from 158.0 mM) within the first 24 hr.

Taken together, these data shown in FIGS. 19–23 demonstrate thatincreases in both the serum calcium and sodium occur after transfer ofpreadult anadromous fish from freshwater to seawater. Moreover, theoverall expression of PVCR protein increases in specific cells involvedin this osmoregulatory response such as intestine. Since PVCRs arecapable of responding to alterations of both calcium and sodium withinthese concentrations ranges, these data indicate that a new “set point”for PVCR activity is established after transfer of fish to seawater.

The data shown in FIGS. 21–23 demonstrate that treatment of preadultanadromous fish with APS Process I causes increases in the serum calciumconcentrations of the fish that mimic those produced by their transferfrom freshwater to seawater. Exposure of the fish to the combination ofcalcium and magnesium in the water and NaCl in the feed causes increasedcalcium intake that mirrors the drinking of hypertonic seawater withoutthe accompanying osmotic stress. Thus, the PVCRs in the APS Process Ifish have been “preconditioned” by their exposure to calcium andmagnesium and, as a result, the fish is more readily able to adapt toseawater when it is subsequently transferred to it.

Anadromous Fish Exhibiting Visible Symptoms of Stress After Transfer toSeawater Possess Elevated Serum Values of Calcium and/or Magnesium. TheInability of Fish to Excrete these Ions is the Major Cause for theirDeath After Seawater Transfer:

When pre-adult anadromous fish are transferred to seawater eitherdirectly from freshwater or after exposure to the APS Process I, someportion of the total number of fish are often unable to adapt to thedramatic differences in osmolality and ionic composition betweenfreshwater and seawater and die of resulting electrolyte imbalances.Observations that include tracking of fish that will ultimately expirewithin a short time interval (24–120 hr) after seawater transferdemonstrates that they begin to exhibit visible signs of high levels ofstress including alterations in their normal light silver bodycoloration to a darker duskier hue as well as displaying of bizarreswimming behavior or markedly decreased activity levels 24–72 hr beforetheir death.

Comparison of serum calcium, magnesium and sodium concentrations fromcontrol nonstressed fish vs fish exhibiting signs of high levels ofstress show that serum ion concentrations in stressed fish aresignificantly higher as compared to control (Table 12).

TABLE 12 Comparison of serum concentrations of juvenile Atlantic salmonand trout in seawater judged by visual inspection as either nonstressedor stressed fish. Serum Concentrations in mM Calcium Magnesium IndustryStandard Juvenile Trout Nonstressed Fish 4.03 ± 0.71 (n = 49) Not DoneStressed Fish 4.58 ± 0.78** (n = 63) Not Done APS Treated AtlanticSalmon Nonstressed Fish 3.74 ± 0.52 2.40 ± 0.77 (n = 15) Stressed fish3.97 ± 0.66 4.07 + 0.60** (n = 16) **P < 0.01

These signs of high stress are directly referable to abnormally elevatedconcentrations of calcium, magnesium and sodium ions within the bodyfluids of the fish. Thus, preadult anadromous fish that are unable toexcrete excess divalent cations as well as process seawater to replacebody water that is lost via osmosis die from the consequences ofelectrolyte imbalances. Anadromous fish below the critical size are notable to rapidly adapt to the new osmotic environment of seawater and dieas a result.

Exposure of Preadult Atlantic Salmon Fish Below the “Critical Size” asDefined by Present Day Industry Standard Methods to the APS Process IIPrevents the Lethal Elevations of Serum Calcium, Magnesium and Sodiumand thus Allows Successful Seawater Transfer of Fish Possessing VerySmall Body Weights:

Pre-adult Atlantic salmon of the St John/St John strain were begun onthe APS Process II including a water mixture (3 mM Ca2+ and 1 mM Mg2+)as well as feed containing a combination of 7% NaCl and 2 gm/kg (w/w)L-Tryptophan (APS Process II) for a total of 49 days while being exposedto a continuous photoperiod. These small, but treated pre-adult Atlanticsalmon (termed APS Process II smolts) were then placed into seawaterinto either a single ocean netpen or into laboratory tanks (15.6° C.)within the research facility. FIG. 24 compares the body characteristicsof APS Process II smolts that adapted successfully to seawater vs APSProcess II smolts from the same group that were unable to adapt toseawater and died.

As shown in FIG. 24, only those Atlantic salmon preadult fish treatedwith the APS Process II with the smallest body weights (approximately10%) experienced post seawater mortalities after 5 days into laboratorytanks. Comparison of the average body size of 90% surviving APS ProcessII smolts vs. those 10% APS Process II smolts that died shows thatunsuccessful APS Process II smolts possessed smaller body weights(5.10+2.2 gm) as compared to average body size of whole APS Process IItransfer group (11.5+5.65 gm). Thus, the critical size for these APSProcess II smolts is approximately 13 gm. This critical body size isonly 13–18.6% (13/70–100) that of the critical size defined previouslyby industry standard techniques. Thus, these data show that the use ofthe Process H has reduced the “critical size” of Atlantic salmonparr/smolt by over 80%.

Quantitation of serum calcium, magnesium and sodium concentrations inAPS Process II smolts that have successfully made the transition fromcalcium/magnesium water mixture to seawater is shown in FIG. 25. It isnoteworthy that serum concentrations of calcium, magnesium or sodium didnot change despite the fact that the average body size of these APSProcess II smolts is less than 20% (11.5/60.5 gm) of the normal“critical size” for Atlantic Salmon smolts produced using present dayindustry standard methods. FIG. 25 shows that neither serum calcium,magnesium or sodium concentrations increase dramatically as would beexpected from data shown in FIG. 20 and Table 2 as well as datapublished previously. Treatment of industry standard Atlantic salmonsmolt/parr with APS Process II results in no dramatic increases in theconcentrations of any ions measured above despite the significantlysmaller body size of APS Process II smolts, as compared to largeindustry standard smolts. Comparison of data shown in FIGS. 22A–B(Industry standard S1 smolts), FIG. 23 (Industry standard S1 smoltstreated with APS Process I vs FIG. 25 (preadult salmon less than 20% ofthe industry standard critical size) also reveals that these serumconcentrations for the smaller APS Process II smolts are comparable tothose displayed by the larger industry standard S1 smolts. In summary,these data show that preadult Atlantic salmon treated with APS ProcessII do not exhibit dramatic changes in their body composition of calcium,magnesium and sodium despite their significantly smaller size. This lackof alterations in the concentrations of these ions greatly reducesstress in these fish and permits them to adapt to seawater readily.

Example 8 The Feed

There are two general methods to prepare feed for consumption by fish aspart of APS Process I and II. These two processes involve eitherreformulation of feed or addition of a concentration solution forabsorption by the feed followed by a top dressing for palatability. Thisdisclosure describes the methodology to prepare feed using each of these2 methods.

Methods:

Feed Manufacture for Salmon Experiments

To reformulate feed, the ingredients are as follows: Base Diet was madeusing the following ingredients and procedure: 30% Squid (liquefied inblender), 70% Corey Aquafeeds flounder diet (powderized in blender).Ingredients were blended into a semi moist “dough” ball. Otheringredients including NaCl or PVCR active compounds were blended intothe base diet by weight according to what the experiment called for.

Moore Clark standard freshwater salmonid diet (sizes 1.2, 1.5. 2.0, 2.5,and 3.5 mm) can also be used. A top dressing was applied to the pelletssuch that top dressing is composed of 4% of the weight of the Base Diet.Top dressing is composed of 50% krill hydrolysate (Specialty MarineProducts Ltd.) and 50% Menhaden fish oil. The top dressing is added forpalatability and sealing of added ingredients.

Other ingredients can include NaCl, MgCl2, CaCl2 or L-Tryptophan thatare added by weight to the base diet by weight.

Preparation of Feed Containing 7% (Weight/Weight) NaCl:

For the APS Process I: Solid sodium chloride or NaCl apportioned at aratio of 7% of the weight of the Moore Clark standard freshwatersalmonid diet weight was added to a volume of tap water approximately3–4 times the weight of NaCl. The mixture was heated to 60–70° C. withmixing via use of a magnetic stirring bar to dissolve salt. The NaClsolution was then poured into a hand held sprayer and applied to theMoore Clark standard freshwater salmonid diet that is tumbling inside ofa 1.5 cubic meter motorized cement mixer. After absorption of the NaClrich solution, the wetted Moore Clark standard freshwater salmonid dietis spread out thinly on window screening and placed in an enclosed racksystem equipped with a fan and 1500 watt heater to expedite dryingprocess. After drying for approximately 6 hr, the dried NaCl-richpellets are returned to the cement mixer and a top dressing is applied.The feed is stored at room temperature until use.

Preparation of Feed Containing 7% (weight/weight) NaCl+PVCR Agonist(Tryptophan) For the APS Process II: Solid sodium chloride or NaClapportioned at a ratio of 7% of the weight of the Moore Clark standardfreshwater salmonid diet weight was added to a volume of tap waterapproximately 3–4 times the weight of NaCl. The mixture was heated to60–70° C. with mixing via use of a magnetic stirring bar to dissolvesalt. USP Grade L-Tryptophan was added to the water at either 2 grams or4 grams for every kg of Moore Clark standard freshwater salmonid dietdepending on formulation need. Dilute hydrochloric acid was added to thewater with mixing until the tryptophan was dissolved and the pH ofsolution was approximately 4.0. The NaCl+Tryptophan solution was thenpoured into a hand held sprayer and was then applied to the Moore Clarkstandard freshwater salmonid diet tumbling inside a cement mixer. Afterabsorption of the NaCl+Tryptophan solution, the wetted Moore Clarkstandard freshwater salmonid diet is then spread out thinly on windowscreening and placed in an enclosed rack system equipped with a fan and1500-watt heater to expedite drying process. After drying forapproximately 6 hr, the dried NaCl/Tryptophan-rich pellets are thenreturned to the cement mixer and a top dressing is applied. The feed isstored at room temperature until use.

Example 9 DNA and Putative Protein Sequences from Partial Genomic Clonesof Polyvalent Cation Receptor Protein Amplified by PCR from the DNA of 8Species of Anadromous Fish

These data provide the partial genomic sequences derived from the PVCRgene in 8 species of anadromous fish. Each of these nucleotide sequencesis unique and thus could be used as a unique probe to isolate thefull-length cDNA from each species. Moreover, this DNA fragment couldform the basis for a specific assay kit(s) for detection of PVCRexpression in various tissues of these fish.

The PVCR has been isolated in several species of salmon, char and trout.Sequences of mammalian CaRs together with the nucleotide sequence ofSKCaR (FIGS. 28A–B) were used to design degenerate oligonucleotideprimers to highly conserved regions in the extracellular domain ofpolyvalent cation receptor proteins using standard methodologies (See GM Preston, Polymerase chain reaction with degenerate oligonucleotideprimers to clone gene family members, Methods in Mol. Biol. Vol. 58Edited by A. Harwood, Humana Press, pages 303–312, 1993). Using theseprimers, cDNA or genomic DNA from various fish species representingimportant commercial products are amplified using standard PCRmethodology. Amplified bands are then purified by agarose gelelectrophoresis and ligated into appropriate plasmid vector that istransformed into a bacterial strain. After growth in liquid media,vectors and inserts are purified using standard techniques, analyzed byrestriction enzyme analysis and sequenced where appropriate. Using thismethodology, nucleotide sequences were amplified.

To generate the data displayed in FIGS. 26 and 27, DNA was isolated frommuscle samples of each of the species indicated using standard publishedtechniques. DNA was then amplified using polymerase chain reaction (PCR)methodology including 2 degenerate PCR primers (DSK-F3 and DSK-R4; SEQID NO:22 and 23). Amplified DNAs were then purified by agarose gelelectrophoresis, subcloned into plasmid vectors, amplified, purified andsequenced using standard methods.

FIG. 26 shows an aligned genomic DNA sequences of 594 nucleotides for 8anadromous fish species, each of which codes for an identical region ofthe PVCR protein. Note that each nucleotide sequence derived from eachspecific species is unique. However, alterations in the DNA sequences ofthese genes often occur at common specific nucleotides within eachsequence of 594 nucleotides.

FIG. 27 shows aligned corresponding predicted protein sequences derivedfrom genomic nucleotide sequences displayed in FIG. 26. Note that only 3alterations in the amino acid sequence of this portion of the PVCR occuras a consequence of alterations in the nucleotide sequence as shown inFIG. 26. All of these changes (Ala to Val; Arg to Lys; and Cys to Tyr)are known as “conservative” substitutions of amino acids in that theypreserve some combination of the relative size, charge andhydrophobicity of the peptide sequence.

All cited references, patents, and patent applications are incorporatedherein by reference in their entirety. Also, companion patentapplication Ser. No. 09/687,373, entitled “Growing Marine Fish in FreshWater,” filed on Oct. 12, 2000; patent application Ser. No. 09/687,476,entitled “Methods for Raising Pre-adult Anadromous Fish,” filed on Oct.12, 2000; patent application Ser. No. 09/687,372, entitled “Methods forRaising Pre-adult Anadromous Fish,” filed on Oct. 12, 2000; ProvisionalPatent Application No. 60/240,392, entitled “Polyvalent Cation SensingReceptor Proteins in Aquatic Species,” filed on Oct. 12, 2000;Provisional Patent Application No. 60/240,003, entitled “PolyvalentCation Sensing Receptor Proteins in Aquatic Species,” filed on Oct. 12,2000, are all hereby incorporated by reference in their entirety.Additionally, application Ser. No. 09/162,021, filed on Sep. 28, 1998,International PCT application No. PCT/US97/05031, filed on Mar. 27,1997, and application Ser. No. 08/622,738 filed Mar. 27, 1996, allentitled, “Polycation Sensing Receptor in Aquatic Species and Methods ofUse Thereof” are all hereby incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes can be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

1. An aquatic food composition for consumption by anadromous fish in freshwater comprising a concentration of NaCl between about 10,000 mg/kg and about 100,000 mg/kg and a PVCR modulator in an amount sufficient to increase expression and/or sensitivity of at least one PVCR in the fish upon consumption of the composition.
 2. The aquatic food composition of claim 1, wherein the PVCR modulator is L-Tryptophan.
 3. The aquatic food composition of claim 2, wherein the L-Tryptophan is in an amount of between about 0.2% and about 0.4% by weight.
 4. The aquatic food composition of claim 2, wherein the L-Tryptophan is in an amount of about 0.2% by weight.
 5. The aquatic food composition of claim 2, wherein the L-Tryptophan is in an amount of between about 0.4% by weight.
 6. The aquatic food composition of claim 1, wherein the NaCl is in a concentration of about 7% by weight.
 7. An aquatic food composition for consumption by anadromous fish in freshwater comprising a concentration of NaCl between about 10,000 mg/kg and about 100,000 mg/kg, a PVCR modulator in an amount sufficient to increase expression and/or sensitivity of at least one PVCR in the fish upon consumption of the composition, and a top dressing that includes fish oil.
 8. The aquatic food composition of claim 7, wherein the top dressing includes at least about 50% by weight of fish oil.
 9. The aquatic food composition of claim 8, wherein the top dressing includes menhaden fish oil.
 10. The aquatic food composition of claim 7, wherein the top dressing includes at least a portion of the PVCR modulator.
 11. The aquatic food composition of claim 7, wherein the top dressing includes at least a portion of the NaCl.
 12. An aquatic food composition for consumption by anadromous fish in freshwater comprising: NaCl in an amount of about 7% by weight; and L-Tryptophan in an amount of about 0.2% by weight; wherein the expression and/or sensitivity of at least one PVCR is increased in the fish upon consumption of the composition.
 13. The aquatic food composition of claim 12, further comprising a top dressing that includes at least about 50% by weight of fish oil.
 14. The aquatic food composition of claim 13, wherein the top dressing includes at least a portion of the L-Tryptophan.
 15. The aquatic food composition of claim 13, wherein the top dressing includes at least a portion of the NaCl.
 16. An aquatic food composition for consumption by anadromous fish in freshwater comprising: NaCl in an amount of about 7% by weight; and L-Tryptophan in an amount of about 0.4% by weight wherein the expression and/or sensitivity of at least one PVCR is increased in the fish upon consumption of the composition.
 17. The aquatic food composition of claim 16, further comprising a top dressing that includes at least about 50% by weight of fish oil.
 18. The aquatic food composition of claim 17, wherein the top dressing includes at least a portion of the L-Tryptophan.
 19. The aquatic food composition of claim 17, wherein the top dressing includes at least a portion of the NaCl. 